U.S. patent number 9,365,607 [Application Number 14/511,821] was granted by the patent office on 2016-06-14 for synthesis of deuterated ribo nucleosides, n-protected phosphoramidites, and oligonucleotides.
This patent grant is currently assigned to ASED, LLC. The grantee listed for this patent is ASED, LLC. Invention is credited to Suresh C. Srivastava, Amy Yasko.
United States Patent |
9,365,607 |
Srivastava , et al. |
June 14, 2016 |
Synthesis of deuterated ribo nucleosides, N-protected
phosphoramidites, and oligonucleotides
Abstract
The present invention is directed towards the synthesis of high
purity deuterated sugars, deuterated phosphoramidites, deuterated
nucleobases, deuterated nucleosides, deuterated oligonucleotides,
and deuterated RNA's of defined sequences which can exhibit
biochemically useful and biologically valuable properties, thus
having potential for therapeutic uses.
Inventors: |
Srivastava; Suresh C.
(Burlington, MA), Yasko; Amy (Bethel, ME) |
Applicant: |
Name |
City |
State |
Country |
Type |
ASED, LLC |
Bethel |
ME |
US |
|
|
Assignee: |
ASED, LLC (Bethel, ME)
|
Family
ID: |
56100439 |
Appl.
No.: |
14/511,821 |
Filed: |
October 10, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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13563343 |
Jul 31, 2012 |
8859754 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07B
59/005 (20130101); C07H 21/02 (20130101); C12N
15/111 (20130101); C12N 2320/51 (20130101); C12N
2310/317 (20130101); C12N 2320/52 (20130101); C12N
2310/11 (20130101) |
Current International
Class: |
C07H
21/02 (20060101) |
Other References
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nucleic acids", 12:3:99-101, (Mar. 2006). cited by applicant .
Sinhababu, A. et al, "Mechanism of action of
S-adenosyl-L-homocysteine hydrolase. Measurement of kinetic isotope
effects using adenosine-3'-d and S-adenosyl-L-homocysteine-3'd as
substrates", J. Am. Chem. Soc., 107:7628-7632, (1985). cited by
applicant .
Moriarity, R. et al, "The specifically 2-monodeuterated
2-deoxy-D-riboses (2(S)-and
2(R)-Deuterio-2-deoxy-D-erthropentoses)", J. Am. Chem. Soc.,
93:12:3/86/3087, (Jun. 16, 1971). cited by applicant .
Wong, M. et al, 2-Deoxypentoses. Stereoselective reduction of
ketene dithioacetals, J. Am. Chem. Soc., 100:11:3548-3553, (May 24,
1978). cited by applicant .
Dupre, M. et al, "Synthese D'adenosine monodeuteriee
stereospecifiquement EN C-5'", Tetrahedron Letters, 31:2783-2786,
(Jun. 5, 1978). cited by applicant .
Roy, S. et al, "New enzymatic synthesis of
2'-deoxynucleoside-2',2'-d2 and the determination of sugar ring
flexibility by solid-state deuterium NMR", J. Am. Chem. Soc.,
108:1675-1678, (1986). cited by applicant .
Pathak, T. et al, "Synthesis of 2'-deoxy-2'(S)-deuterio and
2'-deoxy-2'(R)-deuterio-.beta.-D-nucleosides", Tetrahedron,
42:19:5427-5441, (1986). cited by applicant .
Wu, J. et al, "Regiospecific synthesis of 2'-deoxy-2',2'-dideuterio
nucleosides", Tetrahedron, 43:10:2355-2368, (1987). cited by
applicant .
Hiyama, Y. et al, "Solid-state 2H NMR study of thymidine. Base
rigidity and ribose ring flexibility in deoxynucleosides", J. Am.
Chem. Soc., 111:8609-8613, (Feb. 9, 1989). cited by applicant .
Reed, M. et al, "Acridine- and cholesterol-derivatized solid
supports for improved synthesis of 3'-mofdified oligonucleotides",
Bioconjugate Chem. 2:217-225, (Mar. 22, 1991). cited by applicant
.
Saison-Behmoaras, T. et al, "Short modified antisense
oligonucleotides directed against Ha-ras point mutation induce
selective cleavage of the mRNA and inhibit T24 cells
proliferation", The EMBO Jrnl., 10:5:1111-1118, (1991). cited by
applicant .
Lagos-Quintana, M. et al, "Identification of novel genes coding for
small expressed RNAs", Science, 294:26:853-858, (Oct. 26, 2001).
cited by applicant .
Foldesi, A. et al, "Synthesis of partially-deuterated
2'-deoxyribonucleoside blocks and their incorporation into an
Oligo-DNA for simplification of overcrowding and selective
enhancement of resolution and sensitivity in the 1H-NMR spectra",
Tetrahedron, 54:14487-14514, (Sep. 30, 1998). cited by applicant
.
Chirakul, P. et al, "Stereospecific syntheses of 3'-deuterated
pyrimidine nucleosides and their site-specific incorporation into
DNA", American Chemical Society, Organic Letters, 5:6:917-919,
(Feb. 21, 2003). cited by applicant .
Chen, T. et al, "Synthesis of 3'-deuterated pyrimidine nucleosides
via stereoselective reduction of protected 3-oxoribose",
Tetrahedron Letters, 39:1103-1106, (Nov. 17, 1997). cited by
applicant .
Kinoshita, T. et al, "Preparation of deuterium-labeled nucleosides
by platinum-catalyzed exchange and reduction", Journal of Labelled
Compounds and Radiopharmaceuticals, 9:4:525-534, (Aug. 17, 1981).
cited by applicant.
|
Primary Examiner: Lewis; Patrick
Attorney, Agent or Firm: McHale & Slavin, P.A.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
In accordance with 37 CFR 1.76, a claim of priority is included in
an Application Data Sheet filed concurrently herewith. Accordingly,
the present invention claims priority as a continuation-in-part of
U.S. patent application Ser. No. 13/563,343, entitled, "Synthesis
of Deuterated Ribo Nucleosides, N-Protected Phosphoramidites, and
Oligonucleotides", filed Jul. 31, 2012, now U.S. Pat. No.
8,859,754, issued Oct. 14, 2014. The contents of the above
referenced application are incorporated herein by reference in its
entirety.
Claims
What is claimed is:
1. A modified ribo-oligonucleotide having the formula comprising:
##STR00004## wherein D is deuterium; B is a nucleobase; n is a
number greater than 1 and represents the number of nucleosides in
the oligonucleotide; W is oxygen or sulfur; Y is oxygen, C1-C18
alkoxy, C1-18 alkyl; NHR3 with R3 being C1-C18 alkyl or C1-C4
alkoxy-C1-C6-alkyl; NR3R4 in which R3 is C1-C18 alkyl or C1-C4
alkoxy-C1-C6-alkyl and R4 is C1-C18 alkyl, or in which R3 is C1-C18
alkyl or C1-C4 alkoxy-C1-C6-alkyl and R4 is C1-C18-alkyl, or in
which R3 and R4 form together with the nitrogen atom carrying them,
a 5-6 membered heterocyclic ring which can additionally contain
another hetero atom from the series O, S and N; said modified
ribo-oligonucleotide having at least one phosphate group having a 5
prime to 5 prime linkage, wherein at least one phosphate group
links the 5 prime carbon of one nucleoside to the 5 prime carbon of
a second, independent nucleoside.
2. The modified ribo-oligonucleotide according to claim 1 wherein W
is oxygen and Y is oxygen.
3. The modified ribo-oligonucleotide according to claim 1 wherein W
is sulfur and Y is oxygen.
4. The modified ribo-oligonucleotide according to claim 1 wherein
at least one deuterium is replaced by a hydrogen.
5. The modified ribo-oligonucleotide according to claim 1 wherein B
is partially or fully deuterated.
6. The modified ribo-oligonucleotide according to claim 1 wherein
said nucleotide has a phosphorothioate linkage.
7. The modified ribo-oligonucleotide according to claim 1 wherein
said ribose sugars are partially deuterated.
8. The modified ribo-oligonucleotide according to claim 1 having a
mixture of fully deuterated ribose sugars and partially deuterated
ribose sugars.
9. The modified ribo-oligonucleotide according to claim 1 wherein
the nucleotide sequence is one of SEQ ID NO: 4-SEQ ID NO: 42.
10. The modified ribo-oligonucleotide according to claim 9 wherein
the Y is replaced with X--C--(Y.sub.1Y.sub.2Y.sub.3Y.sub.4),
wherein W can be oxygen (O.sup.-) or sulfur (S.sup.-); Y can be
singly or multiply hydrogen, methyl, ethyl; X can be an electron
attracting group.
11. A modified ribo-oligonucleotide having the formula comprising:
##STR00005## wherein D is deuterium; B is a nucleobase; n is a
number greater than 1 and represents the number of nucleosides in
the oligonucleotide; W is oxygen or sulfur; Y is oxygen, C1-C18
alkoxy, C1-18 alkyl; NHR3 with R3 being C1-C18 alkyl or C1-C4
alkoxy-C1-C6-alkyl; NR3R4 in which R3 is C1-C18 alkyl or C1-C4
alkoxy-C1-C6-alkyl and R4 is C1-C18 alkyl, or in which R3 is C1-C18
alkyl or C1-C4 alkoxy-C1-C6-alkyl and R4 is C1-C18-alkyl, or in
which R3 and R4 form together with the nitrogen atom carrying them,
a 5-6 membered heterocyclic ring which can additionally contain
another hetero atom from the series O, S and N; and said modified
ribo-oligonucleotide having at least one phosphate group having a 3
prime to 3 prime linkage, wherein at least one phosphate group
links the 3 prime carbon of one nucleoside to the 3 prime carbon of
a second, independent nucleoside.
12. The modified ribo-oligonucleotide according to claim 11 wherein
W is oxygen and Y is oxygen.
13. The modified ribo-oligonucleotide according to claim 11 wherein
W is sulfur and Y is oxygen.
14. The modified ribo-oligonucleotide according to claim 11 wherein
at least one deuterium is replaced by a hydrogen.
15. The modified ribo-oligonucleotide according to claim 11 wherein
B is partially or fully deuterated.
16. The modified ribo-oligonucleotide according to claim 11 wherein
said nucleotide has a phosphorothioate linkage.
17. The modified ribo-oligonucleotide according to claim 11 wherein
said ribose sugars are partially deuterated.
18. The modified ribo-oligonucleotide according to claim 11 having
a mixture of fully deuterated ribose sugars and partially
deuterated ribose sugars.
19. The modified ribo-oligonucleotide according to claim 11 wherein
the nucleotide sequence is one of SEQ ID NO: 4-SEQ ID No: 42.
20. The modified ribo-oligonucleotide according to claim 11 wherein
the Y is replaced with X--C--(Y.sub.1Y.sub.2Y.sub.3Y.sub.4),
wherein W can be oxygen (O.sup.-) or sulfur (S.sup.-); Y can be
singly or multiply hydrogen, methyl, ethyl; X can be an electron
attracting group.
21. A modified ribo-oligonucleotide having the formula comprising:
##STR00006## wherein D is deuterium; B is a nucleobase; n is a
number greater than 1 and represents the number of nucleosides in
the oligonucleotide; X is a detecting molecule; W is oxygen or
sulfur; Y is oxygen, C1-C18 alkoxy, C1-18 alkyl; NHR3 with R3 being
C1-C18 alkyl or C1-C4 alkoxy-C1-C6-alkyl; NR3R4 in which R3 is
C1-C18 alkyl or C1-C4 alkoxy-C1-C6-alkyl and R4 is C1-C18 alkyl, or
in which R3 is C1-C18 alkyl or C1-C4 alkoxy-C1-C6-alkyl and R4 is
C1-C18-alkyl, or in which R3 and R4 form together with the nitrogen
atom carrying them, a 5-6 membered heterocyclic ring which can
additionally contain another hetero atom from the series O, S and
N.
22. The modified ribo-oligonucleotide according to claim 21 having
the formula comprising: ##STR00007## wherein X1, X2, and X3 are
detecting molecules.
23. The modified ribo-oligonucleotide according to claim 21 wherein
X1, X2 and X3 are the same detecting molecules.
24. The modified ribo-oligonucleotide according to claim 21 wherein
at least one of X1, X2 and X3 are different detecting
molecules.
25. The modified ribo-oligonucleotide according to claim 21 wherein
said detecting molecules are chromophores or flurophores.
26. The modified ribo-oligonucleotide according to claim 22 wherein
said different detecting molecules are chromophores or
flurophores.
27. The modified ribo-oligonucleotide according to claim 22 wherein
W is oxygen and Y is oxygen.
28. The modified ribo-oligonucleotide according to claim 22 wherein
W is sulfur and Y is oxygen.
29. The modified ribo-oligonucleotide according to claim 22 wherein
at least one deuterium is replaced by a hydrogen.
30. The modified ribo-oligonucleotide according to claim 22 wherein
B is partially or fully deuterated.
31. The modified ribo-oligonucleotide according to claim 22 wherein
the nucleotide sequence is one of SEQ ID NO: 4-SEQ ID NO: 42.
32. The modified ribo-oligonucleotide according to claim 22 wherein
the Y is replaced with X--C--(Y.sub.1Y.sub.2Y.sub.3Y.sub.4),
wherein W can be oxygen (O.sup.-) or sulfur (S.sup.-); Y can be
singly or multiply hydrogen, methyl, ethyl; X can be an electron
attracting group.
33. The modified ribo-oligonucleotide according to claim 22 wherein
said modified ribo-oligonucleotide comprises at least one phosphate
group having a 5 prime to 5 prime linkage, wherein at least one
phosphate group links a 5 prime carbon of one nucleoside to a 5
prime carbon of a second, independent nucleoside.
34. The modified ribo-oligonucleotide according to claim 22 wherein
said modified ribo-oligonucleotide comprises at least one phosphate
group having a 3 prime to 3 prime linkage, wherein at least one
phosphate group links a 3 prime carbon of one nucleoside to a 3
prime carbon of a second, independent nucleoside.
35. A modified ribo-oligonucleotide having the formula comprising:
##STR00008## wherein D is deuterium; B is a nucleobase; L is a
ligand, n is a number greater than 1 and represents the number of
nucleosides in the oligonucleotide; W is oxygen or sulfur; Y is
oxygen, C1-C18 alkoxy, C1-18 alkyl; NHR3 with R3 being C1-C18 alkyl
or C1-C4 alkoxy-C1-C6-alkyl; NR3R4 in which R3 is C1-C18 alkyl or
C1-C4 alkoxy-C1-C6-alkyl and R4 is C1-C18 alkyl, or in which R3 is
C1-C18 alkyl or C1-C4 alkoxy-C1-C6-alkyl and R4 is C1-C18-alkyl, or
in which R3 and R4 form together with the nitrogen atom carrying
them, a 5-6 membered heterocyclic ring which can additionally
contain another hetero atom from the series O, S and N.
36. A modified ribo-oligonucleotide having the formula according to
claim 35 wherein L is cyanoethyl phosphate-polyethylene
glycols.sup.v, where v is a number greater than 1 and represents
the number of glycol units.
37. A modified ribo-oligonucleotide having the formula according to
claim 35 wherein L is cyanoethyl phosphate having a linker attached
with cholesterol, biotin, fluoresceins, cyanine dyes, psoralen,
tetramethylrhodamine dye, dabcyl dye, C-3 disulfide, C-6 disulfide,
symmetrical and asymmetrical hydrocarbon chain (C.sub.2-C.sub.50),
symmetrical and asymmetrical hydrocarbon chain (C.sub.2-C.sub.50)
with a terminal amino group protected with CF3C(.dbd.O) or
phthalamido or FMOC, (C.sub.1-C.sub.16)alkylene-amine protected
with a amine protecting group, (C.sub.1-C.sub.5)alkylene-amine
protected with an azide group; (C.sub.1-C.sub.5)alkylene-amine
protected amine protected with an acetylene (C triple bond CH)
group.
38. The modified ribo-oligonucleotide having the formula according
to claim 35 wherein L is cyanoethyl phosphate-ethane-2-ol-protected
with DMT group or other acid labile groups, cyanoethyl
phosphate-propane -3-ol-protected with DMT group or other acid
labile groups.
39. The modified ribo-oligonucleotide having the formula according
to claim 35 wherein L is (C.sub.1-C.sub.50) alkylene with a
terminal hydroxyl.
40. The modified ribo-oligonucleotide having the formula according
to claim 35 wherein L is lipids, carboxyl groups, or peptide.
41. The modified ribo-oligonucleotide having the formula according
to claim 35 wherein L is a branched phosphoramidite.
42. The modified ribo-oligonucleotide having the formula according
to claim 35 wherein said modified ribo-oligonucleotide comprises at
least one phosphate group having a 5 prime to 5 prime linkage,
wherein at least one phosphate group links a 5 prime carbon of one
nucleoside to a 5 prime carbon of a second, independent
nucleoside.
43. The modified ribo-oligonucleotide having the formula according
to claim 35 wherein said modified ribo-oligonucleotide comprises at
least one phosphate group having a 3 prime to 3 prime linkage,
wherein at least one phosphate group links a 3 prime carbon of one
nucleoside to a 3 prime carbon of a second, independent
nucleoside.
44. The modified ribo-oligonucleotide having the formula according
to claim 35 wherein at least one nucleobase includes a detecting
molecule.
45. The modified ribo-oligonucleotide according to claim 35 wherein
W is oxygen and Y is oxygen.
46. The modified ribo-oligonucleotide according to claim 35 wherein
W is sulfur and Y is oxygen.
47. The modified ribo-oligonucleotide according to claim 35 wherein
at least one deuterium is replaced by a hydrogen.
48. The modified ribo-oligonucleotide according to claim 35 wherein
B is partially or fully deuterated.
49. The modified ribo-oligonucleotide according to claim 35 wherein
said nucleotide has a phosphorothioate linkage.
50. The modified ribo-oligonucleotide according to claim 35 wherein
said ribose sugars are partially deuterated.
51. The modified ribo-oligonucleotide according to claim 35 having
a mixture of fully deuterated ribose sugars and partially
deuterated ribose sugars.
52. The modified ribo-oligonucleotide according to claim 35 wherein
the nucleotide sequence is one of SEQ ID NO: 4-SEQ ID NO: 42.
53. The modified ribo-oligonucleotide according to claim 35 wherein
the Y is replaced with X--C--(Y.sub.1Y.sub.2Y.sub.3Y.sub.4),
wherein W can be oxygen (O.sup.-) or sulfur (S.sup.-); Y can be
singly or multiply hydrogen, methyl, ethyl; X can be an electron
attracting group.
54. A modified ribo-oligonucleotide having the formula comprising:
##STR00009## wherein D is deuterium; B1 or B2 is a nucleobase; n is
a number greater than 1 and represents the number of nucleosides in
the oligonucleotide; W is oxygen or sulfur; Y is oxygen, C1-C18
alkoxy, C1-18 alkyl; NHR3 with R3 being C1-C18 alkyl or C1-C4
alkoxy-C1-C6-alkyl; NR3R4 in which R3 is C1-C18 alkyl or C1-C4
alkoxy-C1-C6-alkyl and R4 is C1-C18 alkyl, or in which R3 is C1-C18
alkyl or C1-C4 alkoxy-C1-C6-alkyl and R4 is C1-C18-alkyl, or in
which R3 and R4 form together with the nitrogen atom carrying them,
a 5-6 membered heterocyclic ring which can additionally contain
another hetero atom from the series O, S and N; n2 is a number
greater than 1 and represents the number of deoxynucleosides in the
oligonucleotide.
55. The modified ribo-oligonucleotide according to claim 54 wherein
W is oxygen and Y is oxygen.
56. The modified ribo-oligonucleotide according to claim 54 wherein
W is sulfur and Y is oxygen.
57. The modified ribo-oligonucleotide according to claim 54 wherein
at least one deuterium is replaced by a hydrogen.
58. The modified ribo-oligonucleotide according to claim 54 wherein
B is partially or fully deuterated.
59. The modified ribo-oligonucleotide according to claim 54 wherein
said nucleotide has a phosphorothioate linkage.
60. The modified ribo-oligonucleotide according to claim 54 wherein
said ribose sugars are partially deuterated.
61. The modified ribo-oligonucleotide according to claim 54 having
a mixture of fully deuterated ribose sugars and partially
deuterated ribose sugars.
62. The modified ribo-oligonucleotide according to claim 54 wherein
the nucleotide sequence is one of SEQ ID NO: 4-SEQ ID NO: 42.
63. The modified ribo-oligonucleotide according to claim 54 wherein
the Y is replaced with X--C--(Y.sub.1Y.sub.2Y.sub.3Y.sub.4),
wherein W can be oxygen (O.sup.-) or sulfur (S.sup.-); Y can be
singly or multiply hydrogen, methyl, ethyl; X can be an electron
attracting group.
64. The modified ribo-oligonucleotide according to claim 54 wherein
said modified ribo-oligonucleotide comprises at least one phosphate
group having a 5 prime to 5 prime linkage, wherein at least one
phosphate group links a 5 prime carbon of one nucleoside to a 5
prime carbon of a second, independent nucleoside.
65. The modified ribo-oligonucleotide according to claim 54 wherein
said modified ribo-oligonucleotide comprises at least one phosphate
group having a 3 prime to 3 prime linkage, wherein at least one
phosphate group links a 3 prime carbon of one nucleoside to a 3
prime carbon of a second, independent nucleoside.
66. The modified ribo-oligonucleotide according to claim 54 further
including at least one detecting molecule.
67. The modified ribo-oligonucleotide according to claim 54 further
including at least one ligand.
68. The modified ribo-oligonucleotide according to claim 54 wherein
B2 is deuterated thymadine.
69. The modified ribo-oligonucleotide according to claim 54 wherein
B2 is undeuterated thymadine.
70. A modified ribo-oligonucleotide having the formula comprising:
##STR00010## wherein D is deuterium; B is a nucleobase; n is a
number greater than 1 and represents the number of nucleosides in
the oligonucleotide; W is oxygen or sulfur; Y is oxygen, C1-C18
alkoxy, C1-18 alkyl; NHR3 with R3 being C1-C18 alkyl or C1-C4
alkoxy-C1-C6-alkyl; NR3R4 in which R3 is C1-C18 alkyl or C1-C4
alkoxy-C1-C6-alkyl and R4 is C1-C18 alkyl, or in which R3 is C1-C18
alkyl or C1-C4 alkoxy-C1-C6-alkyl and R4 is C1-C18-alkyl, or in
which R3 and R4 form together with the nitrogen atom carrying them,
a 5-6 membered heterocyclic ring which can additionally contain
another hetero atom from the series O, S and N; said modified
ribo-oligonucleotide having at least one phosphate group having a 2
prime to 5 prime linkage, wherein at least one phosphate group
links the 2 prime carbon of one nucleoside to the 5 prime carbon of
a second, independent nucleoside.
71. The modified ribo-oligonucleotide according to claim 70 wherein
W is oxygen and Y is oxygen.
72. The modified ribo-oligonucleotide according to claim 70 wherein
W is sulfur and Y is oxygen.
73. The modified ribo-oligonucleotide according to claim 70 wherein
at least one deuterium is replaced by a hydrogen.
74. The modified ribo-oligonucleotide according to claim 70 wherein
B is partially or fully deuterated.
75. The modified ribo-oligonucleotide according to claim 70 wherein
said nucleotide has a phosphorothioate linkage.
76. The modified ribo-oligonucleotide according to claim 70 wherein
said ribose sugars are partially deuterated.
77. The modified ribo-oligonucleotide according to claim 70 having
a mixture of fully deuterated ribose sugars and partially
deuterated ribose sugars.
78. The modified ribo-oligonucleotide according to claim 70 wherein
the nucleotide sequence is one of SEQ ID NO: 4-SEQ ID No: 42.
79. The modified ribo-oligonucleotide according to claim 70 wherein
the Y is replaced with X--C--(Y.sub.1Y.sub.2Y.sub.3Y.sub.4),
wherein W can be oxygen (O.sup.-) or sulfur (S.sup.-); Y can be
singly or multiply hydrogen, methyl, ethyl; X can be an electron
attracting group.
80. The modified ribo-oligonucleotide according to claim 70
including at least one detecting molecule.
81. The modified ribo-oligonucleotide according to claim 70
including at least one ligand.
82. The modified ribo-oligonucleotide according to claim 70
including at least one detecting molecule.
83. The modified ribo-oligonucleotide according to claim 70
including at least one ligand.
84. The modified ribo-oligonucleotide according to claim 1
including at least one detecting molecule.
85. The modified ribo-oligonucleotide according to claim 1
including at least one ligand.
86. The modified ribo-oligonucleotide according to claim 21
including at least one detecting molecule.
87. The modified ribo-oligonucleotide according to claim 21
including at least one ligand.
88. A modified ribo-oligonucleotide having the formula comprising:
##STR00011## wherein D is deuterium; B is a nucleobase; n is a
number greater than 1 and represents the number of nucleosides in
the oligonucleotide; W is oxygen or sulfur; Y is oxygen, C1-C18
alkoxy, C1-18 alkyl; NHR3 with R3 being C1-C18 alkyl or C1-C4
alkoxy-C1-C6-alkyl; NR3R4 in which R3 is C1-C18 alkyl or C1-C4
alkoxy-C1-C6-alkyl and R4 is C1-C18 alkyl, or in which R3 is C1-C18
alkyl or C1-C4 alkoxy-C1-C6-alkyl and R4 is C1-C18-alkyl, or in
which R3 and R4 form together with the nitrogen atom carrying them,
a 5-6 membered heterocyclic ring which can additionally contain
another hetero atom from the series O, S and N; wherein the
nucleotide sequence is one of SEQ ID NO: 13-SEQ ID NO: 42.
Description
FIELD OF THE INVENTION
The present invention relates to oligonucleotides and
oligonucleotide synthesis; and more particularly, to modified RNA,
phosphoramidites, and RNA oligonucleotides, and processes for
synthesizing RNA containing partially or fully saturated deuterated
sugar and/or nucleobases and deuterated phosphoramidites for
synthesis of the modified oligonucleotides.
BACKGROUND OF THE INVENTION
The present invention is directed towards the synthesis of high
purity deuterated sugars, deuterated nucleobases, deuterated
nucleosides and deuterated RNA's of defined sequences which can
exhibit biochemically useful and biologically valuable properties,
thus having potential for therapeutic uses. The past several
decades have seen the development of many RNA and DNA sequences for
use in therapeutics, diagnostics, drug design, selective inhibition
of an RNA sequence within cellular environments, and blocking a
function of different types of RNA present inside the cell. One
approach has been the use of antisense technology. Antisense
oligonucleotides are useful for specifically inhibiting unwanted
gene expression in mammalian cells. Antisense oligonucleotides can
be used to hybridize to and inhibit the function of an RNA,
typically a messenger RNA, by activating RNase H. Primarily, the
oligonucleotides affect the level of the target RNA by activation
of RNase H, which cleaves the RNA strand of DNA/RNA hybrids. As a
result, antisense oligonucleotides have been proposed for the
treatment of diseases. While such technology has the potential to
be a powerful tool for all diseases, several issues, including
molecule stability, have prevented the technology from being a
major disease fighting therapy.
Another approach focuses on silencing gene expression at the mRNA
level with nucleic acid-based molecules. RNA interference (RNAi)
offers great potential for selective gene inhibition and provides
great promise for control and management of various biochemical and
pharmacological processes. Early studies illustrated that RNA
interference in C. elegans is mediated by 21 and 22 nucleotide RNA
sequences, see Fire et al., Nature, 391, 806-811, 1998. This was
further confirmed by studies illustrating the general phenomenon of
specific inhibition of gene expression by small double stranded
RNA's mediated by 21 and 22 nucleotide RNA's, Genes Dev., 15,
188-200, 2001. Simultaneous studies confirmed such phenomenon of
specific gene expression by small double stranded (dS) RNAs in
invertebrates and vertebrates alike. Various studies have also
illustrated the use of RNAi as a powerful tool for selective and
specific gene inhibition and regulation, see Nishikura, K., Cell,
107, 415-418, 2001; Nykanen, et al., Cell, 107, 309-321, 2001;
Tuschl, T., Nat. Biotechnol., 20, 446-448, 2002; Mittal, V., Nature
Rev., 5, 355-365, 2004; Proc. Natl. Acad. Sci. USA, 99, 6047-6052,
2002; Donze, O. & Picard, D., Nucl. Acids. Res., 30, e46, 2002;
Sui, G et al., Natl. Acad. Sci. USA, 99, 5515-5520, 2002; Paddison,
et al., Genes Dev., 16, 948-959, 2002.
In addition to the use of natural double stranded (ds) RNA
sequences, chemically modified RNA have been shown to cause similar
or enhanced RNA interference in mammalian cells using
2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA) into sequences
for siRNA activities, see Dowler, et al., Nucl. Acids Res., 34,
1669-1675, 2006. Various other modifications to improve SiRNA
properties have been pursued, including alterations in backbone
chemistry, 2'-sugar modifications, nucleobase modifications, see
reviews Nawrot, B et al., Med. Chem., 6, 913-925, 2006 and
Manoharan, M. Curr. Opin. Chem. Biol., 8, 570-579, 2004. While
modifications of SiRNA have been tolerated, several studies
indicate an increased toxicity and reduced efficacy see Harborth,
et al., Antisense Nucleic Acid Drug Dev., 13, 83-105, 2003. Chiu et
al. demonstrated that the 2'-O-methyl modification, although
maintaining an A form RNA-like helix, does retain SiRNA activity,
or in some cases, reduces SiRNA activity depending on the number of
such modifications within a sequence, see RNA, 9, 1034-1048, 2003.
It has also been shown that extensive 2'-O methyl modification of a
sequence can be made in the sense strand without loss of SiRNA
activity, see Kraynack, B. A., Baker, B. F., RNA, 12, 163-176,
2006. Bicyclic locked nucleic acids (LNA's) that confer high
binding affinity have been introduced in SiRNA sequences,
especially when the central region of SiRNA sequence is avoided,
see Braash, et al., Biochemistry, 42, 7967-7995, 2003. Similarly,
altritol sugar modified oligonucleotides (ANA), which contain rigid
conformations, and has been shown to faint degradable duplexes with
RNA in a sequence specific manner. In addition, ANAs have been
shown to stay in A (RNA type) conformation. Fisher, M., et al.,
Nucl. Acids Res., 35, 1064-1074, 2007 demonstrated that ANA
modified siRNAs targeting MDR1 gene exhibited improved efficacy as
compared to unmodified controls, specifically effective when
modification was near the 3'-end of sense or anti-sense strand.
Several studies have indicated the potential for siRNA uptake by
various delivery systems. Such delivery systems can then be
exploited in the development of therapeutics.
Cholesterol-conjugated siRNA can achieve delivery into cells and
silence gene expression. In addition, lipid conjugated siRNA, bile
acids, and long chain fatty acids can mediate siRNA uptake into
cells and silence gene expression in vivo. Efficient and selective
uptake of siRNA conjugates in tissues is dependent on the maximum
association with lipoprotein particles, lipoprotein/receptor
interactions and transmembrane protein mediated uptake. High
density lipoproteins direct the delivery of siRNA into the liver,
gut, kidney and steroidal containing organs. Moreover, LDL directs
siRNA primarily to the liver. Studies have indicated that the LDL
receptor is involved in the delivery of siRNA. Therefore, it has
been proposed that siRNA can be designed with chemical
modifications to protect against nuclease degradation, abrogate
inflammation, reduce off target gene silencing, and thereby improve
effectiveness for target genes. Delivery vehicles or conjugates of
lipids and other lipophilic molecules which allow enhanced cellular
uptake are essential for therapeutic developments. Such siRNAs are
presently being developed for human target validation and
interfering with diseases pathways and developing new frontier for
drug development.
The 3'-end of sense strand of siRNA can be modified and attachment
of ligands is most suited at this end, see for example, Ya-Lin Chiu
and Tariq Rana, RNA, 9, 1034-1048, 2003; M. Manoharan, Curr. Opin.
Chem. Biol, 6, 570-579, 2004; Nawrot, B. and Sipa, K., Curr. Top.
Med. Chem., 6, 913-925, 2006; Scaringe, S., et al. Biotechnol., 22,
326-30, 2004. The introduction of lipophilic or hydrophobic groups
and enhancement of siRNA delivery and optimization of targets has
been addressed and achieved through bioconjugation. Generally the
attachment is performed at the 3'-end of the sense strand, but can
be performed on the 3'-end of the anti-sense strand. The design of
nuclease resistant siRNA has been the subject of intense research
and development in attempts to develop effective therapeutics. Thus
base modifications such as 2-thiouridine, pseudouridine, and
dihydrouridine have illustrated the effect on conformations of RNA
molecules and the associated biological activity, see Sipa et al.,
RNA, 13, 1301-1316, 2007. Layzer, et al., RNA, 10, 766-771, 2004,
illustrated that 2'-modified RNA, especially 2'-fluoro, have great
resistance towards nuclease and are biological active in-vivo.
Dande et al., Med. Chem., 49, 1624-1634, 2006 used 4'-thio modified
sugar nucleosides in combination of 2'-O alkyl modification for
improving siRNA properties and RNAi enhancement. Li et al.,
Biochem. Biophys. Res. Comm., 329, 1026-1030, 2005 and Hall et al.,
Nucl. Acids Res., 32, 5991-6000, 2004 illustrated the replacement
of internucleotide phosphate with phosphorothioate and
boranophosphates of siRNAs in vivo.
In addition to in vivo stability and appropriate modification of
nucleosides, bioconjugation of siRNA molecules, RNA molecules,
aptamers and synthetic DNA molecules require key features for cell
membrane permeability. Insufficient cross-membrane cellular uptake
limits the utility of siRNAs, other single stranded RNAs, or even
various DNA molecules. Thus cholesterol attached at the 3'-end of
siRNA has been shown to improve in vivo cell trafficking and
therapeutic silencing of the gene, see Soutschek et al., Nature,
432, 173-0178, 2004. In addition to cholesterol, various
conjugations have been developed, including natural and synthetic
protein transduction domains (PTDs), also called cell permeating
peptides (CPPs) or membrane permanent peptides (MPPs). PTDs are
short amino acid sequences that are able to interact with the
plasma membrane. The uptake of MPP-siRNA conjugates takes place
rapidly. Such peptides can be conjugated preferably to the 3'-end
of the strand. PEG (polyethylene glycols-oligonucleotide)
conjugates have been used in various conjugate complexes and
possess significant gene silencing effect after uptake in target
cells, see Oishi et al., Am. Chem. Soc., 127, 1624-1625, 2005.
Aptamers have been used for site specific delivery of siRNAs. Given
that aptamers have high affinity for their targets, conjugates with
siRNA act as an excellent delivery system and results in efficient
inhibition of the target gene expression, see Chu et al., Nucl.
Acids Res., 34(10), e73, 2006. These molecules can be conjugated at
the 3'-end of siRNA or other biologically active oligonucleotides.
Various lipid conjugations at the 3'-end can be attached to
oligonucleotides synthesized by the process described by the
invention and can be utilized for efficient internalization of
oligonucleotides. The lipophilic moiety consists of a hydroxyl
function to synthesize a phosphoramidite. Similarly the lipophilic
moiety can have carboxylic function at the terminus. The latter can
be coupled to a 3'-amino group having a spacer, synthesized by last
addition of amino linkers such as C-6 amino linker amidite, of the
reverse synthesized oligonucleotide, to the carboxylic moiety using
DCC (dicyclohexyl cabodiimide) or similar coupling reagent, see
Paula et al., RNA, 13, 431-456, 2007.
Micro-RNA (miRNA) is a large class of non coding RNAs which have
been shown to play a role in gene regulation, see Bartel, D. P.
Cell, 116, 281-297, He et al. Nat. Rev. Genet, 5:522-531, 2004;
Lagos-Quintana et al., Science, 204:853-858, 2001. It is estimated
that there are at least 1000 miRNA scattered across the entire
human genome. Many of these miRNAs have been shown to down regulate
large numbers of target mRNAs, see Lim et al., Nature, 433:769-773,
2005. Different combinations of miRNAs may be involved in
regulation of target gene in mammalian cell. siRNA has been shown
to function as miRNAs, see Krek et al., Nat. Genet., 37: 495-500,
2005; Doench et al., Genes Dev., 17:438-442, 2003. Micro-RNAs have
great potential as therapeutics and in gene regulation, Hammond, S.
M., Trends Mol. Med. 12:99-101, 2006. A vast amount of effort is
currently being devoted towards understanding miRNA pathways, their
role in development and diseases, and their role in cancer.
Additionally, miRNA targets are being developed for therapeutic and
diagnostics development. A great number of miRNA are being
identified and their role is being determined through microarrays,
PCR and informatics. Synthesis of RNA designed to target miRNA also
requires RNA synthesis and similar modification, as required for
SiRNAs, for stability of RNA and bioconjugation resulting in better
cellular uptakes. The instant invention will greatly accelerate the
pace of this research and development.
Synthesis of therapeutic grade RNA and siRNA requires modification
or labeling of the 3'-end of an oligonucleotide. In the case of
siRNA, generally it is the 3'-end of the sense strand. The
synthesis of 3'-end modified RNA requiring lipophilic, long chain
ligands or chromophores, using 3' to 5' synthesis methodology is
challenging, and requires corresponding solid support. Such
synthesis generally results in low coupling efficiency and lower
purity of the final oligonucleotide in general because of a large
amount of truncated sequences containing desired hydrophobic
modification. The authors of the instant invention approached this
problem by developing reverse RNA monomer phosphoramidites for RNA
synthesis in the 5' to 3'-direction. This approach leads to very
clean oligonucleotide synthesis, thus allowing for introduction of
various modifications at the 3'-end cleanly and efficiently.
In order to increase stability, oligonucleotides containing lipids
have been synthesized. Attachment of the lipids provides for
efficient delivery of the RNA and an increase in the cellular
concentration of the oligonucleotides. Hydrophobic molecules, such
as cholesterol, can bind to LDL particles and lipoproteins to
activate a delivery process involving these proteins to transport
oligonucleotides. Lipped nucleic acids may also reduce the
hydrophilicity of oligonucleotides. It has also been shown that
lipidoic nucleic acids improve the efficacy of oligonucleotides,
see Shea, et al., Proc. Natl. Acad. Sci. USA 86, 6553, 1989;
Oberhauser, B., and Wagner, E., Nucleic Acids Res., 20, 533, 1992;
Saison-Behmoaras, et al,. The EMBO Journal, 10, 1111, 1991; Reed et
al., Bioconjugate Chem., 2, 217, 1991; Polushin, et al.,
Nucleosides & Nucleotides, 12, 853, 1993; Marasco et al.,
Tetrahedron Lett., 35, 3029, 1994. A series of hydrophobic groups
such as adamantane, eicosenoic acid, cholesterol, and dihexadecyl
glycerol were attached to oligodeoxy nucleotide sequences at the
3'-end and were hybridized to complementary RNA sequences. The Tm
was found to be unaffected indicating that such groups do not
interfere with oligo hybridization properties see Manoharan et al.,
Tetrahedron Lett., 36, 1995; Manoharan, et al., Tetrahedron Lett.,
36, 3651-3654, 1995; Gerlt, J. A. Nucleases, 2nd Edition, Linn, S.
M., Lloyd, R. S., Roberts, R. J., Eds. Cold Spring Harbor
Laboratory Press, p-10, 1993.
For efficient delivery of synthetic RNA molecules, PEG attachment
to various oligonucleotides has shown favorable properties.
PEG-oligomers have shown enzymatic stability by preventing fast
digestion. The thermal melting behavior was not affected, thereby
retaining properties of double strand formation. Srivastava et al.,
Nucleic Acids Symposium Series, 2008, 52, 103-104 recently
developed a reverse RNA synthesis process for clean attachment of
lipophilic and large molecules to synthetic RNA.
DESCRIPTION OF THE PRIOR ART
Deuterium labeling studies & NMR analysis have been carried out
for many nucleosides and oligonucleotides. The structure and
dynamics of DNA and RNA is vital to understanding their biological
functions. This has been investigated by a variety of
physico-chemical techniques. Amongst these techniques, Nuclear
Magnetic Resonance (NMR) spectroscopy have been utilized
extensively as a powerful tool because it provides conformational
information on the implication of variation of local structures and
the dynamics under a biological condition. This has been refined
using powerful computers and high resonance energy instruments.
With increasing magnetic field, the higher sensitivity reduces the
amount of an oligomer needed to obtain a good quality spectrum, and
increases the dispersion of resonance signals reducing the spectral
complexity due to resonance overlap which results from second order
J couplings to first order.
Most of the studies describing incorporation of deuterium at
specific positions of deoxynucleosides, ribonucleosides, and
modified nucleosides were carried out in an effort to determine the
structure of oligonucleosides and conformational details by proton
Nuclear Magnetic Resonance (NMR). Proton NMR spectrum of
oligonucleotides are generally quite complex and do not reveal
conformational & structural information. As a result of
oligonucleotides having significant overlapping NMR resonance,
structure determination of deuterated oligonucleotides has been
used for NMR structure determination of biologically functional DNA
or RNA molecules. In order to overcome problems associated with
resonance, investigators developed non-uniform deuterium labeling
techniques, see Foldesi et al., J. Tetrahedron, 1992, 48, 9033;
Foldesi et al., J., Biochem. Biophys. Methods, 1993, 26. Deuterium
labeled oligonucleotides simplifies NMR spectras, allowing
determination of both J couplings and NOE volumes in an unambiguous
manner from a small domain of a large molecule see Glemarec et al.,
J. Nucleic Acids Res., 1996, 24, 2002 and Ludwig, J. Acta Biochem.
Biophys Acad Sci., 1981, 16, 131.
Similarly, site specific deuteration of a large number of
oligo-DNAs and RNAs have been used to study NMR structures by the
"NMR-window" concept in which only a small segment of the
oligonucleotide is NMR visible. This approach was used to solve the
NMR structure of a 21-mer RNA hairpin loop, see Nucleic Acids
Research 1996 24:1187 and Nucleosides and Nucleotides 1997,
5&6, 743, and a 31-mer stem-internal loop-stem-internal
loop-stem-hairpin loop RNA. Diastereospecifically C-2' (deuterium
labeled nucleoside block in oligo-DNA (Journal Tetrahedron, 1995,
51, 10065) was successfully utilized in NMR interpretation the
collection of reduced spin-diffusion as well as the extraction of
.sup.3JH1', H2'' and .sup.3JH1', H2'' coupling constants.
Huang et al., Acids Research, 1997, 25, 4758-4763 showed that in
two dimensional (2D) NOESY spectra of oligonucleotides, if H-8 of
purines and H-6 in pyrimidines are replaced with deuterium then the
entire cross peaks correlating the nucleobase with sugar protons
disappear. Similarly researchers have been interested in studying
the role of dynamics of interaction of proteins with DNA by 2H NMR.
Solid state 2D NMR provides valuable information about the movement
of various functional groups in an oligonucleotide. Chirukul and
coworkers have shown that specific deuteration plays a very
significant role in determining such structural features, see
Chirakul, et al., Nucleosides, Nucleotides and Nucleic acids, 2001,
20, 1903-1913.
Enzyme recognition with deuterium substitution in place of hydrogen
or enzymatic binding is not adversely affected. The enzyme
recognition of a particular sequence is the first step in
biochemical interaction of oligonucleotides for their specific
roles, and deuterium labeling does not change the biochemical
process of site recognition. Similarly it is known that
hybridization of a double strand is not effected by deuterium
labeling, since deuterium and hydrogen atomic radii are very close
for any disruption in recognition pattern.
It is expected that multiple covalent labeling of deuterium in
place of hydrogen (carbon-hydrogen bonds to carbon-deuterium bond)
in the sugar portion of an oligonucleotide slows down the rate of
digestion of oligonucleotides which takes place rapidly in cellular
environment with exo and endo nucleases. The quick digestion of the
oligonucleotide is demonstrated by shorter half life of
oligonucleotide and clearance from body. This is much more
pronounced in RNA molecules as compared to DNA molecules. The slow
digestion of a therapeutic oligonucleotide is expected to add extra
advantage to a therapeutic candidate, while other physical or
biochemical properties are not affected. Various biochemical
effects of deuterated ribo-oligonucleotides is anticipated
deuterated oligos are expected to slow digestion of
oligonucleotides to smaller fragments, and have no effect with
respect to hydrogen bonding, RNAse H editing activity, or
recognition by RISC complex. Intracellular hydrolysis or deuterium
exchanges my result in liberation of deuterium oxide
(D.sub.20).
The enzymatic method of deuterium exchange has been carried out
routinely for deuterium labeling. However the exchange method is
not complete due to equilibrium which exists in enzymatic
reactions. It is anticipated that deuterium labeled
oligonucleotides will similarly exchange deuterium with hydrogen
within the cellular environment resulting in release of deuterium
oxide within the cellular environment. Since deuterium oxide is
known as a nutritional agent, oligonucleotides of the instant
invention may provide nutritional value.
The use of deuterium exchange for the spectral assignment of
nucleosides and oligonucleotides has been carried out quite
extensively. Deuteration of the nucleobase residues has been
described in exchange of protons at C8-purine and C5-cytosine with
deuterioammonium bisulfite at pH 7.8 in deoxyoligomers which gave
90-95% atom .sup.2H incorporation. Brush et al. Biochemistry 1988,
27, 115; Brush et al,. Am. Chem. Soc. 1998, 110, 4405 described
platinum-catalyzed exchange at C5-methyl of thymidine in
.sup.2H.sub.2O.
A large variety of enzymatic and chemical methods have been
developed for deuterium incorporation at both sugar and nucleoside
levels to provide high levels of deuterium incorporation (D/H
ratio). The enzymatic method of deuterium exchange generally has
low levels of incorporation and provides significant levels of
stray resonances. Enzymatic incorporation has further complications
due to cumbersome isolation techniques which are required for
isolation of deuterated mononucleotide blocks. Schmidt et al., Ann.
Chem. 1974, 1856; Schmidt et al., Chem. Ber., 1968, 101, 590,
describes synthesis of 5',5''-.sup.2H2-Adenosine which was prepared
from 2',3'-O-isopropylideneadenosine-5'-carboxylic acid or from
methyl-2,3-isopropylidene-.beta.-D-ribofuranosiduronic acid, Dupre,
M. and Gaudemer, A., Tetrahedron Lett. 1978, 2783. Kintanar, et
al., Am. Chem. Soc. 1998, 110, 6367 reported that diastereoisomeric
mixtures of 5'-deuterioadenosine and 5'(R/S)-deuteratedthymidine
can be obtained with reduction of the appropriate 5'-aldehydes
using sodium borodeuteride or lithium aluminum deuteride (98 atom %
.sup.2H incorporation). Berger et al., Nucleoside & Nucleotides
1987, 6, 395 described the conversion of the 5'-aldehyde derivative
of 2'deoxyguanosine to 5' or 4'-deuterio-2'-deoxyguanosine by
heating the aldehyde in .sup.2H.sub.2O/pyridine mixture (1:1)
followed by reduction of the aldehyde with NaBD.sub.4.
Ajmera et al., Labelled Compd. 1986, 23, 963 described procedures
to obtain 4'-Deuterium labeled uridine and thymidine (98 atom %
.sup.2H). Sinhababu, et al., J. Am. Chem. Soc. 1985, 107, 7628)
demonstrated deuterium incorporation at the C3' (97 atom % 2H) of
adenosine during sugar synthesis upon stereoselective reduction of
1,2:5,6-di-O-isopropylidene-B-D-hexofuranos-3-ulose to
1,2:5,6-di-O-isopropylidene-3-deuterio-B-D-ribohexofuranose using
sodium borodeuteride and subsequently proceeding further to the
nucleoside synthesis. Robins, et al., Org. Chem. 1990, 55, 410
reported synthesis of more than 95% atom .sup.2H incorporation at
C3' of adenosine with virtually complete stereoselectivity upon
reduction of the 2'-O-tert-butyldimethylsilyl(TBDMS)
3-ketonucleoside by sodium borodeuteride in acetic acid. David, S.
and Eustache, J., Carbohyd. Res. 1971, 16, 46 and David, S. and
Eustache, J., Carbohyd. Res. 1971, 20, 319 described syntheses of
2'-deoxy-2'(S)-deuterio-uridine and cytidine. The synthesis was
carried out by the use of 1-methyl-2-deoxy-2'-(S)-deuterio
ribofuranoside.
Radatus, et al., J. Am. Chem. Soc. 1971, 93, 3086 described
chemical procedures for synthesizing 2'-monodeuterated (R or
S)-2'-deoxycytidines. These structures were synthesized from
selective 2-monodeuterated-2-deoxy-D-riboses, which were obtained
upon stereospecific reduction of a 2,3-dehydro-hexopyranose with
lithium aluminum deuteride and oxidation of the resulting glycal.
Wong et al. J. Am. Chem. Soc. 1978, 100, 3548 reported obtaining
-Deoxy-1-deuterio-D-erythro-pentose,
2-deoxy-2(S)-deuterio-D-erythro-pentose and
2-deoxy-1,2(S)-dideuterio-D-erythro-pentose from D-arabinose by a
reaction sequence involving the formation and LiAlD.sub.4 reduction
of ketene dithioacetal derivatives.
Pathak et al. J., Tetrahedron 1986, 42, 5427) reported
stereospecific synthesis of all eight 2' or
2''-deuterio-2'-deoxynucleosides by reductive opening of
appropriate methyl 2,3-anhydro-.beta.-D-ribo or
.beta.-D-lyxofuranosides with LiAlD.sub.4. Wu et al. J. Tetrahedron
1987, 43, 2355 described the synthesis of all
2',2''-dideuterio-2'-deoxynucleosides, for both deoxy and
ribonucleosides, starting with oxidation of C2' of sugar and
subsequent reduction with NaBD.sub.4 or LiAlD.sub.4 followed by
deoxygenation by tributyltin deuteride. Roy et al. J. Am. Chem.
Soc. 1986, 108, 1675, reported 2',2''-Dideuterio-2'-deoxyguanosine
and thymidine can be prepared from 2-deoxyribose 5-phosphate using
2-deoxyribose 5-phosphate aldolase enzyme in .sup.2H.sub.20
achieving some 90 atom % deuteration.
Therefore, it is clear that each position of the sugar residue can
be selectively labeled. A number of these deuterated nucleosides
have been used in solid-state .sup.2H-NMR studies on the internal
motions of nucleosides and oligonucleotides, see Hiyama et al. J.
Am. Chem. Soc. 1989, 111, 8609; Alam, T and Drobny, G P.,
Biochemistry, 1990, 29, 3421; Alam et al., Biochemistry, 1990, 29,
9610; Huang et al., J. Am. Chem. Soc. 1990, 112, 9059; Drobny, G P.
et al., Biochemistry, 1991, 30, 9229. In the temperature dependent
line shape analysis in solid-state .sup.2H-NMR spectroscopy, the
stereoselectivity of 2' versus 2'' labeling or the level of
deuteration does not play a significant role. The use of
specifically deuterium labeled nucleotides for the simplification
of 1D and 2D .sup.1H-NMR spectra in solution studies was not very
useful for structural information. However, most extensive use of
deuteration in the 1D NMR studies was performed by Danyluk et al.
These workers isolated pre-deuterated .sup.2H-labeled
mononucleotides (.about.90 atom % .sup.2H incorporation) in a
tedious manner from RNA digest of blue-green algae grown in
.sup.2H.sub.20. These pre-deuterated nucleoside blocks were then
used to obtain a wide variety of partially deuterated dimers and
trimers for the purpose of resonance assignments in 1D .sup.1H-NMR
spectra (200-300 MHz). Synthesis of 4',5',5''-.sup.2H3-adenosine
was carried out and this was coupled to appropriately blocked
adenosine 3-phosphite to give ApA* (pA* 4',5',5''-.sup.2H3-pA).
This dimer allowed the unequivocal measurement of the difference
between phosphorus and H-3' (Kondo et 1., Am. Cem. Soc. 1972, 94,
5121; Kondo, Labeled Compd. 1973, 9, 497; Ezra, et al.,
Biochemistry, 1975, 53, 213; Kondo and Danylik., Biochemistry,
1976, 15, 3627; Lee, et al., Biochemistry, 1976, 15, 3627; Ezra, et
al., Biochemistry, 1977, 16, 1977. Similarly synthesis of
4',5',5''-.sup.2H3-guanosine can be carried out to synthesize
guanosine rich oligonucleotides.
A useful alternative method of stereospecific deuteration was
developed to synthesize polydeuterated sugars. This method employed
exchange of hydrogen with deuterium at the hydroxyl bearing carbon
(i.e. methylene and methine protons of hydroxyl bearing carbon)
using deuterated Raney nickel catalyst in .sup.2H.sub.20. Detailed
studies revealed structure dependent difference in exchange rates,
high level of epimerization, significantly lower extent of
deoxygenation, and difficulties in the reproducibility of the level
of deuteration (Balza et al., Res., 1982, 107, 270; Angyal et al.
Carbohydr. Res. 1986, 157, 83; Koch et al. Res. 1978, 59, 341; Wu
et al. J. Org. Chem. 1983, 48, 1750; and Angyal et al. Res. 1986,
157, 83).
Various techniques are available to synthesize fully deuterated
deoxy and ribonucleosides. Thus in one method, exchange reaction of
deuterated Raney nickel-.sup.2H.sub.20 with sugars, a number of
deuterated nucleosides specifically labeled at 2,3' and 4'
positions were prepared. The procedure consisted of deuteration at
2, 3 and 4 positions of methyl .beta.-D-arabinopyranoside by Raney
nickel-.sup.2H.sub.20 exchange reaction followed by reductive
elimination of 2-hydroxyl group by tributyltin deuteride to give
methyl .beta.-D-2,2',3,4-.sup.2H.sub.4-2-deoxyribopyranoside which
was converted to methyl
.beta.-D-2,2',3,4-.sup.2H4-2-deoxyribofuranoside and glycosylated
to give various 2,2',3,4-.sup.2H4-nucleosides (>97 atom %
.sup.2H incorporation for H3' & H4'; .about.94 atom % .sup.2H
incorporation for H2 and H2') (Pathak, T., Chattopadhyaya, J.
Tetrahedron 1987, 43, 4227; Koch, H. J., Stuart, R. S., Carbohydr.
Res. 1977, 59. C1; Balza, F., Cyr, N., Hamer, G K., Perlin, A. S.,
Koch, H. J., Stuart, R. S., Carbohydr. Res. 1977, 59, C7; Koch, H.
J., Stuart, R. S., Carbohydr. Res. 1978, 64, 127; Koch, H. J.,
Stuart, R. S., Carbohydr. Res. 1978, 59, 341; Balza, F., Perlin, A.
S. Carbohydr. Res., 1982, 107, 270; Angyal, S. J., Odier, L.
Carbohydr. Res., 1983, 123, 13.; Wu, G D., Serianni, A. S., Barker,
R. J., Org. Chem. 1983, 48, 1750; Angyal, S. J., Stevens, J. D.,
Odier, L. Carbohydr. Res. 1986, 157, 83; Kline, P. C., Serianni, A.
S. Magn. Reson. Chem., 1988, 26, 120; Kline, P. C., Serianni, A. S.
Magn. Reson. Chem., 1990, 28, 324; Robins, M. J., Wilson, J. S.,
Hansske, F., J. Am. Chem. Soc. 1983, 105, 4059.
Methyl .beta.-D-erythrofuranoside, when treated with deuterated
Raney Ni, produced methyl
.beta.-D-2,3,4(S)-.sup.2H3-erythrofuranoside (.about.75 atom %
.sup.2H incorporation at C2 and C4(S) positions and 100% atom
.sup.2H incorporation at C3) (Kline, P. C.; Serianni, A. S. Magn.
Reson. Chem., 1988, 26, 120. This sugar was converted to
D-3,4,5(S)-.sup.2H3-ribose. These nucleosides were subsequently
reduced to the corresponding
3',4',5'(S)-.sup.2H3-2'-deoxynucleosides (Koch, H. J.; Stuart, R.
S. Carbohydr. Res. 1978, 64, 127; Kline, P. C., Serianni, A. S.,
Magn. Reson. Chem., 1990, 28, 324). Similar to compound 3',4',5'
(S)-.sup.2H3-ribonucleosides,
1',2',3',4',5',5''(S)-.sup.2H6-ribonucleosides can be synthesized
starting with fully deuterated and appropriately protected
ribose.
SUMMARY OF THE INVENTION
Oligonucleotide based therapeutics is a strong component of
rational drug design approach and a number of oligonucleotides are
currently in the market or at various stages of clinical trials.
Previously, deuterium modified nucleosides have been synthesized at
specific positions of deoxy-sugars and purine and pyrimidine bases.
Deuterated DNA synthons based on phosphotriester technology or
phosphoramidite have been synthesized and utilized for synthesis of
defined sequence oligonucleotides. These studies have been directed
solely for the purpose of conformational studies of DNA and RNA,
determination of active site for enzyme assisted catalytic
reactions. However deuterated oligonucleotides have not been
investigated for therapeutic application in humans or the role
which they can elicit as biological and biochemical agents.
The instant invention describes deuterium labeled phosphoamidites,
ribose units having solid support caps, oligonucleotides, a process
for synthesizing deuterium labeled nucleosides and
oligonucleotides, and a process for synthesizing deuterated
nucleosides and oligonucleotides which contain deuterium ranging
from 0.1% to 98% per position is envisaged. Once a known percentage
of deuterium has been incorporated in the nucleoside, such
nucleosides can further be modified in subsequent steps until the
synthesis of the phosphoramidites or solid support bound
nucleosides for solid phase oligonucleotide synthesis occurs. The
deuterium ratio of 0.1 to 98% in further steps will be maintained.
Such specific and controlled deuteration has not been proposed or
carried out in past to the best of our knowledge. The deuterated
ribo-oligonucleotides formed provide RNA sequences with enhanced
stability.
Deuterium labeling of an oligonucleotide is not expected to present
toxic effects. Selective deuterium modified oligonucleotides either
in selected positions of sugar, purine, pyrimidine bases or total
deuteration of sugar positions and nucleobases will contribute to
the improvement of biological properties of oligonucleotides.
Oligonucleosides specifically deuterated in various positions of
the sugar portion of the ribose are expected to increase enzymatic
stabilities and substantially increase stability of a therapeutic
oligonucleotide. Deuterium substitution is not known to affect the
enzyme recognition or enzymatic binding. Site specific atom
transfer has been utilized for structural information of cleavage
of a specifically deuterium labeled dodecamer, see Voss, et al., J.
Am. Chem. Soc., 1990, 112, 9669-9670. The enzyme recognition of a
particular sequence is the first step in biochemical interaction of
oligonucleotides for their specific roles, and deuterium labeling
does not change the biochemical process of site recognition.
Similarly hybridization of a double strand is not affected by
deuterium labeling.
It is anticipated that deuterium labeled oligonucleotides will not
affect the hydrogen bonding with a complementary strand either by
Watson Crick base pairing mechanism, Hoogsten or other
hybridization mechanisms applicable to DNA/DNA hybridization,
DNA/RNA hybridization, RNA/RNA hybridization. RNAse H cleavage
between deuterated DNA with complementary RNA, which is involved in
anti-sense based oligo-therapeutic approach, is not expected to be
affected by the presence of deuterium covalently attached to the
sugar backbone or nucleobases. Thus deuterium labeled
oligonucleotides should play a role in the anti-sense mode of
therapeutic action. Additionally, it should be possible to develop
deuterated siRNAs for therapeutic application. Based on the various
schemes presented for synthesis of ribonucleosides and
oligonucleotides with one or more deuterium in a nucleoside of an
oligo nucleotide chain, it is anticipated that designing aptamers
with selective or fully deuterated RNA sequences can be
accomplished. The chemical method of synthesis and deuterium
labeling in nucleosides will be done in sugar and nucleobases at
positions which are stable and non-exchangeable in general, such as
at the carbon hydrogen bonds (C--H). However within the cell there
is expected to be slow exchange of deuterium with hydrogen with
slight basic pH. Due to slow release of deuterium by exchange
mechanism in vivo (C-D.fwdarw.C--H), such deuterium labeled
oligonucleotides will offer the advantage of nutritionally
beneficial effects. Deuterium labeled oligonucleotides, therefore,
may have enormous potential to replace therapeutic oligonucleotides
which have natural hydrogen atoms in various non-ionizable
positions of nucleosides and in oligonucleotides. In order to
determine the effect of specific levels of deuteration in
nucleosides of an oligonucleotide, a very low level deuteration
such as 0.1% all the way up to 98% deuteration of a specific
covalent carbon hydrogen bond will be carried out and such
oligonucleotides will be studied for its biochemical and biological
effects and roles. Such systematic biological study will provide
better guidance to development of drugs and therapeutics. Such
studies have not been proposed or carried out to the best of our
knowledge.
As used herein, the term "oligonucleotides" refers to a plurality
of nucleotides joined together in a specific sequence defined by
the natural or modified heterocyclic base moieties. Representative
heterocyclic base moieties include, but are not limited to,
nucleobases such as adenine, guanine, cytosine, uracil, as well as
other non-naturally-occurring and natural nucleobases such as
xanthine, hypoxanthine, 2-aminoadenine, 2,6-diamino purine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine, 5-halo
uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil
(pseudo uracil), 4-thiouracil, 8-halo, oxa, amino, thiol,
thioalkyl, hydroxyl and other 8-substituted adenines and guanines,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine, 7-deazaadenine, 7-deazaguanine. Modified
nucleobases as described herein define synthetic nucleobases or
nucleobases that have been changed from their naturally occurring
state, such as deuteratedadenine, deuteratedcytosine,
deuteratedguanine, and deuterateduracil.
Accordingly, it is a primary objective of the present invention to
teach deuterated nucleosides, phosphoramidites and
oligonucleotides, a process of synthesizing fully deuterated
phosphoramidites and oligonucleotides, and a process of
synthesizing deuterated phosphoramidites and oligonucleotides
containing 0.1%-98% deuterium at various positions.
It is a further objective of the present invention to teach a
process of making derivatized ribo nucleoside and phosphoramidites
with deuterium labeled covalently at various positions of
nucleosides and products made thereof.
It is a further objective of the present invention to teach
ribonucleosides and phosphoramidites with deuterium labeled
covalently at various positions of nucleosides.
It is a still further objective of the present invention to teach
the process of making deuterium labeled oligoribonucleotides with
natural phosphodiester backbone, and products made thereof.
It is a still further objective of the present invention to teach
deuterium labeled oligoribonucleotides with natural phosphodiester
backbone.
It is yet another objective of the present invention to teach the
process of making deuterium labeled oligoribonucleotides with
phosphothioate backbone, and products made thereof.
It is a still further objective of the present invention to teach
deuterium labeled oligoribonucleotides with variant backbones.
It is yet another objective of the present invention to teach
deuterated oligonucleotides having one or more detection molecules,
labels, or tags.
It is a still further objective of the present invention to teach
deuterated oligonucleotides having deoxyribose units attached at
the end of the oligonucleotide.
It is another objective to the present invention to teach
deuterated oligonucleotides having 5 prime to 5 prime
internucloetide linkages.
It is another objective to the present invention to teach
deuterated oligonucleotides having 3 prime to 3 prime
internucloetide linkages.
It is yet another objective of the present invention to teach
deuterium labeled oligoribonucleotides with phosphothioate
backbone.
It is another objective to the present invention to teach
oligonucleotides that have stability enhancing deuterated
backbones.
It is yet another objective of the present invention to teach
deuterated oligonucleotides useful for therapeutic treatments.
It is another objective to the present invention to teach
deuterated RNA antisense oligonucleotides useful for therapeutic
treatments.
Other objects and advantages of this invention will become apparent
from the following description taken in conjunction with any
accompanying drawings wherein are set forth, by way of illustration
and example, certain embodiments of this invention. Any drawings
contained herein constitute a part of this specification and
include exemplary embodiments of the present invention and
illustrate various objects and features thereof.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A is a chemical structure of a modified nucleobase,
illustrated as deuteratedadenine.
FIG. 1B is a chemical structure of a modified nucleobase,
illustrated as deuteratedguanine.
FIG. 1C is a chemical structure of a modified nucleobase,
illustrated as deuteratedcytosine.
FIG. 1D is a chemical structure of a modified nucleobase,
illustrated as deuterateduracil;
FIG. 2 illustrates Scheme 1, synthesis of
1-O-Acetate-.alpha./.beta.2,3,5-O-tribenzoyl-1-2,3,4,5,5'
pentadeuterium-D ribofuranoside;
FIG. 3 illustrates Scheme 2,
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-2',3',4',5',5''penta
deuterium 3'-Cyanoethyl n,n-diisopropyl phosphoramidite-.beta.-D
ribofuranosyl-Uridine;
FIG. 4 illustrates Scheme 3, synthesis of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-succinyl
Icaa-CPG-2',3',4',5',5'' penta deuterium .beta.-D ribofuranosyl)
Uridine;
FIG. 5 illustrates Scheme 4, synthesis of
5'-O-dimetoxytrityl-2'-O-terbutyldimethyl Silyl-3'-N, N-diisopropyl
cyanoethyl phosphoramidite-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-N.sup.4 benzoyl Cytidine;
FIG. 6 illustrates Scheme 5, synthesis of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-succinyl
Icaa-CPG-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-N.sup.4 benzoyl Cytidine;
FIG. 7 illustrates Scheme 6, synthesis of an alternative embodiment
of a modified phosphoramidite in accordance with the instant
invention, illustrated as 5'-O-dimethoxytrityl-2',3',4',5',5''
penta deuterium-.beta.-D ribofuranosyl-N.sup.6 benzoyl
adenosine;
FIG. 8 illustrates Structure C1, a representative illustration of a
particular embodiment of a deuterated oligonucleotide in accordance
with the instant invention;
FIG. 9A illustrates Structure D1, an alternative embodiment of the
deuterated oligonucleotide having a phosphodiester internucleotide
linkage;
FIG. 9B illustrates Structure D2, an alternative embodiment of the
deuterated oligonucleotide having a phosphate backbone variant,
illustrated as phosphorothioate internucleotide linkage;
FIG. 9C illustrates an embodiment of the deuterated oligonucleotide
having at least one 3 prime to 3 prime linkage;
FIG. 9D is an embodiment of the deuterated oligonucleotide having
at least one 5 prime to 5 prime linkage;
FIG. 9E illustrates an embodiment of the deuterated oligonucleotide
having at a detection member, label, or tag;
FIG. 9F illustrates an embodiment of the deuterated oligonucleotide
having at a plurality of detection members, labels, or tags;
FIG. 9G illustrates an embodiment of the deuterated oligonucleotide
having at least one ligand;
FIG. 9H illustrates an embodiment of the deuterated oligonucleotide
having at least one ligand and one or more detection members,
labels, or tags;
FIG. 9I illustrates an embodiment of the deuterated oligonucleotide
having a deoxyribose end cap;
FIG. 9J illustrates a 3'-DMT-deuterated nucleoside;
FIG. 9K illustrates a 3'-DMT-deuterated
nucleoside-5'-succinyl-spacer solid support;
FIG. 9L illustrates a general scheme of the synthesis of deuterated
nucleosides-3'-DMT-5'-solid supports;
FIG. 9M illustrates 3'-DMT-deuterated nucleoside-5'-phosphoramidite
which may be sued in the 5' to 5' oligonucleotide linkage;
FIG. 9N illustrates a chromophore bearing modified deuterated
nucleoside phosphoramidite;
FIG. 9O illustrates an additional example of a chromophore bearing
modified deuterated nucleoside phosphoramidite;
FIG. 9P illustrated synthesis scheme for deuterated
thymidine-3'-solid support and deuterated thymidine
phosphoramidite;
FIG. 9Q-A is an embodiment of the deuterated oligonucleotide having
at least one 2 prime to 5 prime linkage;
FIG. 9Q-B is an embodiment of the deuterated oligonucleotide having
at least one 2 prime to 5 prime linkage, with detecting
molecules;
FIG. 9R illustrates a deuterium monomer amidites for 2',5' linked
oligonucleotides;
FIG. 9S illustrates a synthesis scheme of deuterium monomer
amidites for 2',5' linked oligonucleotides;
FIG. 10 is a summary chart of an HPLC analysis of the deuterated
nucleosides and phosphoramidites, using a Shimazdu, Model, HPLC
Column: Chromsep SS(4.6.times.250 mm) with Chrosep Guard column
Omnisphere 5 C18;
FIG. 11A is 1H-NMR spectrum of 1-O-Acetate-.alpha./.beta.2,3,5
tribenzoyl ribofuranoside;
FIG. 11B is a positive ion mss spectrum of
1-O-Acetate-.alpha./.beta.2,3,5 tribenzoyl ribofuranoside; Lot#:
SK38-38, Calculated mass: 504.14; Observed Mass: 522.40;
FIG. 12A is a 1H-NMR spectrum of
1-O-Acetate-.alpha./.beta.2,3,5-tribenzoyl-2,3,4,5,5'
pentadeuterium-D ribofuranoside (structure VI), with the H-4 proton
shown at approx. 50% intensity, thereby indicating approx. 50%
deuterium incorporation at this position;
FIG. 12B is a positive ion mass spectrum of
1-O-Acetate-.alpha./.beta.2,3,5-tribenzoyl-1-2,3,4,5,5'
pentadeuterium-D ribofuranoside (VI); Calculated mass: 509.17;
Observed Mass: 526.80 (M+ Sodium);
FIG. 13A is a HPLC chromatogram of
2',3',5'-tri-hydroxy-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-Uridine (structure IX);
FIG. 13B is a HPLC report of 2',3',5'-tri-hydroxy-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl-Uridine (structure IX);
FIG. 13C is a mass spectrum of 2',3',5'-tri-hydroxy-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl-Uridine (structure IX);
Calculated mass: 249.10; Observed Mass: 247.30;
FIG. 14A is a HPLC report of 5'-O-dimethoxy trityl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl-Urdine (Structure X);
FIG. 14B is 1H-NMR spectrum of 5'-O-dimethoxy
trityl-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-Uridine (Structure X);
FIG. 14C is a 1H-NMR spectrum of 5'-O-dimethoxy
trityl-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-Uridine (Structure X);
FIG. 14D is a 1H-NMR spectrum of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-O-succinyl
pyridinium salt-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-N.sup.4 benzoyl Cytidine (compound structure
XXIII);
FIG. 15A is a HPLC chromatogram of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl-Uridine (structure XI);
FIG. 15B is a HPLC chromatogram of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl-Uridine; (structure XI);
FIG. 15C is a 1H-NMR spectrum of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl-Uridine; (structure XI);
FIG. 15D is a mass spectrum of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-2',3',4',5',5''penta
deuterium .beta.-D ribofuranosyl-Uridine (structure XI); Calculated
mass: 665.32; Observed Mass: 664.00;
FIG. 16A is a HPLC chromatogram of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-N,N-diisopropyl
cyanoethyl phosphoramidite-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl Uridine (structure XIII);
FIG. 16B is HPLC report of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-N,N-diisopropyl
cyanoethyl phosphoramidite-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl Uridine (structure XIII); Purity: 96.72%;
FIG. 16C is a UV analysis of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-N,N-diisopropyl
cyanoethyl phosphoramidite-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl Uridine (structure XIII);
FIG. 16D is a UV analysis report of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-N,
N-diisopropyl cyanoethyl phosphoramidite-2',3',4',5',5'' penta
deuterium .beta.-D ribofuranosyl Uridine (structure XIII);
FIG. 16E is a mass spectrum of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-N,N-diisopropyl
cyanoethyl phosphoramidite-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl Uridine (structure XIII); Calculated mass: 865.43;
Observed Mass: 866.50; (Mass+Sodium Ion (888.4);
FIG. 16F is a .sup.31P NMR spectrum of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-N,
N-diisopropyl cyanoethyl phosphoramidite-2',3',4',5',5'' penta
deuterium .beta.-D ribofuranosyl Uridine (structure XIII); Lot#:
SK188-38. sharp doublet at 150.560 & 150.069 ppm; purity: 100%;
.DELTA.=0.491;
FIG. 17A is a HPLC chromatogram of
5'-O-dimetoxytrityl-2'-O-terbutyldimethyl Silyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl-N.sup.4 Benzoyl Cytidine
(structure XIV);
FIG. 17B is a HPLC report of
5'-O-dimetoxytrityl-2'-O-terbutyldimethyl Silyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl-N.sup.4 Benzoyl Cytidine
(structure XIV);
FIG. 17C is a 1H-NMR spectrum of
5'-O-dimetoxytrityl-2'-O-terbutyldimethyl Silyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl-N.sup.4 Benzoyl Cytidine
(structure XIV);
FIG. 17D is a mass spectrum of
5'-O-dimetoxytrityl-2'-O-terbutyldimethyl Silyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl-N.sup.4 Benzoyl Cytidine
(structure XIV); Calculated mass: 768.36; Observed Mass:
769.30;
FIG. 17E is a 1H-NMR spectrum of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-O-succinyl
pyridinium salt-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-Uridine (structure XIV);
FIG. 17F is a positive mode-mass spectrum of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-O-succinyl
pyridinium salt-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-Uridine (structure XIV); Calculated mass: 764.83;
Observed Mass: 788.10 (+Sodium Ion);
FIG. 18A is a HPLC report of 2',3',5'-tri Hydroxy-2',3',5', 5''
penta deuterium .beta.-D ribofuranosyl-N.sup.4 benzoyl Cytidine
(structure XVIII);
FIG. 18B is a 1H-NMR spectrum of 2',3',5'-tri Hydroxy-2',3',5', 5''
penta deuterium .beta.-D ribofuranosyl-N.sup.4 benzoyl Cytidine
(structure XVIII);
FIG. 18C is a mass spectrum of 2',3',5'-tri Hydroxy-2',3',5', 5''
penta deuterium .beta.-D ribofuranosyl-N.sup.4 benzoyl Cytidine
(structure XVIII); Calculated mass: 352.14; Observed Mass:
352.50;
FIG. 18D is a mass spectrum of 2',3',5'-tri Hydroxy-2',3',5', 5''
penta deuterium .beta.-D ribofuranosyl-N.sup.4 benzoyl Cytidine
(structure XVIII); Calculated mass: 352.14; Observed Mass:
352.50;
FIG. 19A is a positive mode-mass spectrum of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-O-succinyl
pyridinium salt-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-Uridine (structure XIV); Calculated mass: 764.83;
Observed Mass: 788.10 (+Sodium Ion);
FIG. 19B is a HPLC report of 5'-O-dimethoxytrityl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl-N.sup.4 Benzoyl Cytidine
(compound XIX);
FIG. 19C is a 1H-NMR spectrum of
5'-O-dimethoxytrityl-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-N.sup.4 Benzoyl Cytidine (compound XIX);
FIG. 19D is a 1H-NMR spectrum of
5'-O-dimethoxytrityl-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-N.sup.4 Benzoyl Cytidine (compound XIX);
FIG. 20A is a HPLC chromatogram of
5'-O-dimetoxytrityl-2'-O-terbutyldimethyl Silyl 3'-N, N-diisopropyl
cyanoethyl phosphoramidite-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-N.sup.4 benzoyl Cytidine (structure XXII); Purity:
78.88%;
FIG. 20B is a: HPLC report of
5'-O-dimetoxytrityl-2'-O-terbutyldimethyl Silyl-3'-N, N-diisopropyl
cyanoethyl phosphoramidite-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-N.sup.4 benzoyl Cytidine (structure XXII); Purity:
78.88%;
FIG. 20C is a UV analysis of
5'-O-dimetoxytrityl-2'-O-terbutyldimethyl Silyl-3'-N, N-diisopropyl
cyanoethyl phosphoramidite-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-N.sup.4 benzoyl Cytidine (structure XXII);
FIG. 20D is a UV analysis of
5'-O-dimetoxytrityl-2'-O-terbutyldimethyl Silyl-3'-N, N-diisopropyl
cyanoethyl phosphoramidite-2',3',4',5', 5'' penta deuterium
.beta.-D ribofuranosyl-N.sup.4 benzoyl Cytidine (structure
XXII);
FIG. 20E is a .sup.31P NMR spectrum of
5'-O-dimetoxytrityl-2'-O-terbutyldimethyl Silyl-3'-N, N-diisopropyl
cyanoethyl phosphoramidite-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-N.sup.4 benzoyl Cytidine (structure XXII); sharp
doublet at 150.576 & 149.852 ppm; Purity: 95%;
.DELTA.=0.724;
FIG. 20F is a .sup.31P NMR spectrum of
5'-O-dimetoxytrityl-O-terbutyldimethyl Silyl-3'-N, N-diisopropyl
cyanoethyl phosphoramidite-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-N.sup.4 benzoyl Cytidine (structure XXII); sharp
doublet at 150.576 & 149.852 ppm; Purity: 95%; A=0.724;
FIG. 21A is a.sup.31P NMR spectrum of
5'-O-dimetoxytrityl-2'-O-terbutyldimethyl Silyl-3'-N, N-diisopropyl
cyanoethyl phosphoramidite-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-N.sup.4 benzoyl Cytidine (structure XXII); sharp
doublet at 150.576 & 149.852 ppm; Purity: 95%;
.DELTA.=0.724;
FIG. 21B is a mass spectrum of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-O-succinyl
pyridinium salt-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl-N.sup.4 benzoyl Cytidine (compound structure XXIII);
Calculated mass: 867.95; Observed Mass: 869.20;
FIG. 22A is a HPLC chromatogram of 5'-O-dimethoxy
trityl-2'3',4',5',5''-penta deuterium .beta.-D
ribofuranosyl-N.sup.6 benzoyl Adenosine (structure XXVIII);
FIG. 22B is a HPLC chromatogram of 5'-O-dimethoxy
trityl-2'3',4',5',5''-penta deuterium .beta.-D
ribofuranosyl-N.sup.6 benzoyl Adenosine (structure XXVIII);
FIG. 22C mass spectrum of 5'-O-dimethoxy
trityl-2'3',4',5',5''-penta deuterium ribofuranosyl-N.sup.6 benzoyl
Adenosine (structure)(XVIII); Lot #09015RDV Calculated mass:
678.28; Observed Mass: 679.2;
FIG. 23A is a capillary electrophoresis analysis of the purified
oligonucleotide of SEQ ID No.1, fully deuterated RNA;
FIG. 23B is a capillary electrophoresis report of the purified
oligonucleotide SEQ ID No.1, fully deuterated RNA;
FIG. 23C is a UV analysis of the purified oligonucleotide SEQ ID
No.1, fully deuterated RNA;
FIG. 24A is a capillary electrophoresis analysis of the purified
oligonucleotide SEQ ID No.2, approx. 25% deuterated RNA;
FIG. 24B is a capillary electrophoresis report of the purified
oligonucleotide SEQ ID No.2, approx. 25% deuterated RNA;
FIG. 24C is a UV analysis of the purified oligonucleotide SEQ ID
No.2, approx. 25% deuterated RNA;
FIG. 25A is a capillary electrophoresis analysis of the purified
oligonucleotide SEQ ID No.3 natural RNA;
FIG. 25B is a capillary electrophoresis report of the purified
oligonucleotide SEQ ID No.3 natural RNA;
FIG. 25C is a UV analysis of the purified oligonucleotide SEQ ID
No.3 natural RNA;
FIG. 26A is a capillary electrophoresis analysis of the purified
oligonucleotide SEQ ID No.4 natural RNA;
FIG. 26B is a capillary electrophoresis report of the purified
oligonucleotide SEQ ID No.4 natural RNA;
FIG. 26C is a UV analysis of the purified oligonucleotide SEQ ID
No.4 natural RNA;
FIG. 27A is a capillary electrophoresis analysis of the purified
oligonucleotide SEQ ID No.5 natural RNA;
FIG. 27B is a capillary electrophoresis report of the purified
oligonucleotide SEQ ID No.5 natural RNA; and
FIG. 27C is a UV analysis of the purified oligonucleotide SEQ ID
No.5 natural RNA.
DETAILED DESCRIPTION OF THE INVENTION
The instant invention describes high purity deuterated ribose and
sugars, deuterated ribose-based nucleotides, deuterated RNA
oligonucleotides, and controlled processes for synthesizing
deuterium incorporated oligonucleotides for use in therapeutics.
The controlled process would entail a method of development for
various selected deuteration ranging from 0.1% to 98%, and
analytical methods to ascertain the reaction conditions. The
synthesis process provides deuterated oligonucleotides containing
deuterium ranging from 0.1% per position to 98% per position. After
incorporation of deuterium in varying percentages within
nucleoside, further chemical synthesis will be performed to produce
phosphoramidites which will maintain the percent deuterium at each
step till the step of phosphoramidite. Subsequently such fixed
ratio D/H oligonucleotide synthons will be used to produce
oligonucleotide. Once the percent incorporation of deuterium has
been determined by various analytical methods such as proton NMR
and mass spectroscopy, the ratio of deuterium/hydrogen will not be
affected if proper choice of reaction conditions is maintained. The
instant invention further describes the selected examples
controlled synthesis of deuterium labeled nucleoside-3'-succinate
nucleosides with partial or full saturation of deuterium label
which varies from 0.1%-98% deuterium at specific positions of the
sugar and purine/pyrimidine bases for use in solid phase
oligonucleotide synthesis. Therefore, the instant oligonucleotide
synthesis process is carried out similar to conventional
oligonucleotide synthesis, i.e. from the 3'-end to 5'-end
direction.
The deuterated ribose and sugars, deuterated ribose-based
nucleotides, deuterated RNA oligonucleotides of the present
invention may therefore be used for therapeutic benefits.
Oligonucleotide therapy, i.e., the use of oligonucleotides to
modulate the expression of specific genes, offers an opportunity to
selectively modify the expression of genes without the undesirable
non-specific toxic effects of more traditional therapeutics. In an
illustrative example, the deuterated ribose and sugars, deuterated
ribose-based nucleotides, deuterated RNA oligonucleotides of the
present invention may be used in antisense therapies. The present
invention therefore may be used to provide a modified antisense RNA
with enhanced protection to provide a more stable, not easily
digested, antisense RNA. The oligonucleotides of the present
invention can therefore be used in clinical practice for any
disease and against any target RNA for which antisense therapy is
now known to be suitable or which is yet to be identified. The
deuterated oligonucleotides of the present invention may be used
for other nucleic based molecule therapies including silencing gene
expression at the mRNA level with nucleic acid-based molecules,
such as RNA interference.
Several illustrative steps for synthesizing deuterium nucleoside,
sugar and base protection, phosphoramidites and the corresponding
oligonucleotide contemplated are described below. Synthesis of
sugar deuterium protected nucleosides involves selective
deuteration of non-exchangeable protons, such as H-1, H-2, H-3, H-4
and H-5, 5' of B-D-ribose. The H-1' and H-4' protons are slightly
acidic in nature when they become part of nucleoside and have the
tendency to get exchanged to a certain extent with hydrogen. As a
result, these two protons do not give greater than 90% D/H ratio.
While the protons H-2', H-3', H-5', 5'' have higher pK, and hence
can be deuterated to greater than 95% of D/H ratio, they do not
readily exchange back to hydrogen in protic medium during reaction
or when in contact with slightly basic pH conditions
The present invention discloses a modified phosphoramidite having
the structure of Structure A:
##STR00001## wherein X or X1 represents deuterium or hydrogen, R1
represents a blocking group, R2 independently represents a blocking
group, R3 is a phosphate protecting group, preferably cyanoethyl
dialkylamino, and R4 is a independently a protecting group,
preferably 3'B-cyanoethyl protecting group, and B represents a
nucleobase. Although 1' position of the ribose sugar is also
deuterated, however the extent of deuterium can be variable at this
position and can be exchanged for H after being deuterated.
Therefore, the 1' position is illustrated as D/H in the Figures.
From our data the deuterium incorporation at this position is
approx. 50:50: deuterium:hydrogen. Therefore in our further
discussions if deuterium incorporation is reduced to a lower
deuterium in our formulations, the deuterium enrichment at 1'
position will become almost 50% of the rest of the position as
compared to deuteration in other positions of ribose ring. The
deuterium at the 4' position could be variable as well.
The blocking, or protecting group, generally renders a chemical
functionality of a molecule inert to specific reaction conditions
and can later be removed from such functionality in a molecule
without substantially damaging the remainder of the molecule. As
part of the oligonucleotide process, functional groups on the
nucleobases and the 2' sugar group can are blocked. Hydroxyl
protecting groups according to the present invention include a wide
variety of groups. Preferably, the protecting group is stable under
basic conditions but can be removed under acidic conditions.
Preferably, R1 (5' hydroxyl group) is dimethoxytrityl (DMT). Other
representative hydroxyl protecting groups include, but are not
limited to trityl, monomethoxytrityl, trimethoxytrityl,
9-phenylxanthen-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl
(Mox). Preferably, R2 (2'hydroxy group) is protected with
t-butyldimethylsilyl (TBDMS). Other groups, such as with
t-butyldimethylsilyloxymethyl (TOM) group may be used as well. The
phosphate protecting group functions to protect the phosphorus
containing internucleotide linkage or linkages during, for example,
solid phase oligonucleotide synthetic regimes. Treatment of the
internucleotide linkage or linkages that have a phosphorus
protecting group thereon with a deprotecting agent, such as aqueous
ammonium hydroxide, will result in the removal of the phosphorus
protecting group and leave a hydroxyl or thiol group in its place.
In addition to those listed above, other protecting groups such as,
but not limited to diphenylsilylethyl, delta.-cyanobutenyl, cyano
p-xylyl (CPX), methyl-N-trifluoroacetyl ethyl (META) and acetoxy
phenoxy ethyl (APOE) group can be used as well.
The nucleobase B may be natural bases, such as adenine, guanine,
cytosine, or uracil. B may also be modified bases, such as
deuteratedadenine, see FIG. 1A, deuteratedguanine, see FIG. 1B,
deuteratedcytosine, see FIG. 1C, deuterateduracil, see FIG. 1D, or
other modified bases known to one of skill in the art, including or
analogs of natural bases, synthetic bases, and modified bases such
as, but not limited, to hypoxanthine (inosine), 5-methylcytosine,
5-azacytosine, 5-halogenated uracil and cytosine, and
5-alky-substituted nucleobases such as C-5 propyne uracil and C-5
propyne cytosine, which have also been deuterated. B may also
contain a blocking group, such as benzoyl protecting group, or
isobutyryl protecting group, acetyl protecting group, phenoxyacetyl
protecting group, 4-isopropylphenoxyacetyl protecting group, or
dimethylformamidino, dimethylacetaminidine protecting group.
FIG. 2 describes the synthesis of an illustrative example of a
starting material in the process of synthesizing deuterated
RNA-nucleosides, n-protected phosphoramidites, and
oligonucleotides. The synthesis of the
1-O-Acetate-.alpha./.beta.2,3,5-O-tribenzoyl-1-2,3,4,5,5'
pentadeuterium-D ribofuranoside, structure VI was carried out
according to the Scheme, starting with .alpha./.beta.-D
ribofuranoside (-D-Ribose; structure I). Deuterium was introduced
by slight modification of the procedure described by A. Foldesi, F.
R. Nilson, C. Glemarec, C. Gioeli & J. Chattopadhyaya,
Tetrahedron, 9033, 1992. Procedure for synthesis of deuterated
Raney Nickel was also adopted from the same authors in the
reference cited here with slight modification to improve the
efficiency of deuterium incorporation. The steps involved synthesis
of 1-O-methyl-.alpha./.beta.-D ribofuranoside (II) from D-Ribose
(I). 1-O-methyl .alpha./.beta.2,3,4,5,5' pentadeuterium-D
ribofuranoside (III) was synthesized from compound II with
deuterated Raney Nickel.
1-O-methyl-.alpha./.beta.2,3,5-tribenzoyl-2,3,4,5,5'
pentadeuterium-D ribofuranoside (IV) was synthesized from compound
having structure III by carrying out benzoylation under mild
conditions. 1-Bromo-.alpha./.beta. D 2,3,5-tribenzoyl-2,3,4,5,5'
pentadeuterium-D ribofuranoside (V) was synthesized from compound
IV first by selective removal of 1-O-methyl group to generate
1-hydroxyl sugar which was subsequently replaced by bromine without
isolation of the intermediate 1-hydroxyl sugar. The compound V was
proceeded directly without purification for the synthesis of
1-O-Acetate-.alpha./.beta. 2,3,5-tribenzoyl-2,3,4,5,5'
pentadeuterium-D ribofuranoside (VI). Compound VI was crystallized
and fully characterized by 1H NMR, see FIG. 12A. The percent
deuterium incorporated at each sugar position was confirmed from
this analysis and the sugar was further characterized by Mass
spectral analysis, see FIG. 12B.
Synthesis of 1-O-Acetate-.alpha./.beta.
2,3,5-O-tribenzoyl-2,3,4,5,5' pentadeuterium-D ribofuranoside,
Scheme 1
Preparation of Deuterium Raney-Nickel Catalyst:
Deionized water, 192 mL was placed in a 500 ml Erlenmeyer flask
equipped with a thermometer and Teflon coated magnetic stirrer. The
Erlenmeyer flask was placed inside a plastic beaker which was half
filled with water and located on top of a hot plate/magnetic
stirrer. Sodium hydroxide pellets (51.2 g was slowly added into the
water within the flask while gently stirring. The gentle stirring
maintained the water temperature to about 50.degree. Celsius (C).
The mixture was stirred until al all the sodium hydroxide (NaOH)
pellets had dissolved. Prior to adding additional chemicals, the
temperature inside flask was maintained at approximately 50.degree.
C. Subsequently raney nickel alloy, (Sigma Aldrich) 40 g was
gradually added in small portions within 30 minute time frame. The
temperature of water outside, i.e. within the beaker, was
maintained at approximately 50.degree. C.+/-4.degree. C. After
addition of the Raney Nickel Alloy, the composition was stirred for
approximately 60 minutes while maintaining the inside temperature.
Subsequently, the reaction flask was cooled down slowly to room
temperature, taking approximately 1 hour. Deionized water was added
to the flask 1 liter at a time and carefully decanted out. This
process was repeated two additional times for a total of 3 times.
During each of the water additions and decanting, all solid
materials was left within the flask. After completion of the 3
water and decanting steps, the solid was transferred to a 500 ml
filtration flask. A tube was connected to the filtration flask to
remove any over flown water created while deionized water was added
to the top of the filtration flask. The contents of the filtration
flask was continuously washed and stirred until all turbidity was
gone. Once the turbidity was gone, additional washing with
deionized water was continued using approximately 20 liters of
deionized water. Washing was terminated upon the water having a pH
6.5-7.0 and the supernatant was clear.
Deuterated Raney Nickel catalyst was subsequently prepared. The
catalyst particles after washing were transferred into a septum
capped bottle. Teflon coated magnetic stirrer was placed in the
bottle and a rubber stopper was placed on top of the septum bottle.
The bottle was purged with Argon. The suspension was stirred for 1
minute, after which the particles were allowed to settle. Water was
carefully removed using Pasteur-pipette. This process was carried
out 4 times, each time requiring addition of by adding 1.5 ml
deionized water, stirring, and careful removal of the water.
Subsequently deuterium oxide (D2O, 1.5 ml; Cambridge Isotope Labs.,
Massachusetts, purity greater than 98%) was added. The mixture was
stirred for 30 minutes. After the solid settled to the bottom, the
liquid was carefully removed by pipette. The process was repeated
two additional times, adding additional deuterium oxide (D.sub.2O,
1.5 ml) and stirring for 30 minutes. Each time the septum capped
bottle was opened and reagents added, the bottle was flushed with
Argon and quickly sealed with septum. After three times, 3 ml of
D.sub.2O was added. Then mixture was stirred for 1 hour, followed
by removal of the supernatant. This process was repeated 12
additional times, each time purging the bottle with Argon. The
mixture was treated with D.sub.2O (10 ml) and kept sealed overnight
after purging with Argon. The supernatant was carefully removed,
followed by addition of fresh D.sub.2O (10 ml) in the same manner.
The supernatant was decanted out.
Synthesis of 1-O-methyl .alpha./.beta. 2,3,4,5,5' pentadeuterium-D
ribofuranoside (Structure III)
To 8 grams of 1-O-methyl-.alpha./.beta.-D ribofuranoside 10 ml of
D2O (10 ml) was added. The solution was evaporated on a rotavapor.
This process was repeated two additional time using 10 ml D2O each
time. The residue was dissolved in 160 ml of D2O. Deuterated Raney
Nickel (40 ml) was transferred into the solution. Argon was bubbled
into the reaction mixture for 10 minutes. The reaction mixture was
then maintained on an oil bath at 110.degree. C. for 7 days under
Argon atmosphere. The reaction mixture was cooled to room
temperature and filtered through a bed of celite and washed with a
small volume of deionized water. The filtrate was evaporated on a
rotavapor. The residue was co-evaporated with pyridine three times,
and dried an additional 6 hours using a direct vacuum line. The
process yielded 6.8 g of oily product.
Synthesis of 1-O-methyl-.alpha./.beta. 2,3,5-tribenzoyl-2,3,4,5,5'
pentadeuterium-D ribofuranoside (Structure IV)
Dried 1-O-methyl-.alpha./.beta.2,3,4,5,5' pentadeuterium-D
ribofuranoside (III, 6.8 g was) was placed in a round bottom three
neck flask and set up with a pressure equalizing funnel and a
magnetic stirrer. Dry distilled dichloromethane (34.1 ml) was
added. The reaction mixture was stirred. Dry pyridine (68.2 ml) was
then added. The solution was stirred at zero degrees Celsius.
Subsequently, benzoyl chloride (21.2 ml) was added drop wise
through pressure equalizing funnel in the sealed reaction flask.
After addition of the benzoyl chloride, the pressure equalizing
funnel was removed and replaced with a stopper. The mixture was
kept in a sealed polyethylene bag at 0-4.degree. C. in a
refrigerator for 48 hours. The reaction was poured on ice and water
mixture and the reaction mixture kept for 1 hour. The gummy
material was extracted with chloroform, washed with chilled
(0-5.degree. C.) saturated sodium bicarbonate solution, followed by
a brine solution. The organic layer passed through anhydrous sodium
sulfate, and the solution was evaporated on a rotavapor. The
residue was subsequently co-evaporated with pyridine, followed by
addition with dry toluene. Further drying, 1 undertaken on direct
vacuum line, was performed for 6 hours. An oily product was
obtained and used to synthesize 1-Bromo-.alpha./.beta.
D-2,3,5-tribenzoyl-2,3,4,5,5' pentadeuterium-D ribofuranoside.
Synthesis of 1-Bromo-.alpha./.beta. D 2,3,5-tribenzoyl-2,3,4,5,5'
pentadeuterium-D ribofuranoside (Structure V)
Toluene dried 1-O-methyl-.alpha./.beta.2,3,5-tribenzoyl-2,3,4,5,5'
pentadeuterium-D ribofuranoside (IV) was dissolved in a solution of
33% hydrogen bromide (HBr) made in glacial acetic acid and sealed
tightly. The solution was stirred at room temperature. After 30
minutes, the reaction mixture was cooled to 8-10.degree. C.
Subsequently, the glacial acetic acid (200 ml) was added to the
reaction mixture. Deionized water (130 ml) was then added in a drop
wise manner. The reaction mixture was stirred for 23 minutes. The
reaction mixture was poured on 5-10.degree. C. cooled deionized
water. The gummy mass was extracted with chloroform. Chilled
(0-5.degree. C.) aqueous sodium bicarbonate solution was added to
the organic layer until the pH of the organic layer was basic (pH
>8). The organic layer was separated and washed with chilled
aqueous sodium bicarbonate solution once again, followed by passing
the organic layer over anhydrous sodium sulfate. The filtered
solution was evaporated on a rotary evaporator. The gummy solid was
co-evaporated with dry pyridine two times. An oily product was
obtained and used in next step.
Synthesis of 1-O-Acetate-.alpha./.beta. 2,3,5-tribenzoyl-2,3,4,5,5'
pentadeuterium-D ribofuranoside (Structure VI)
The dried product, 1-Bromo-.alpha./.beta.
2,3,5-tribenzoyl-2,3,4,5,5' pentadeuterium-D ribofuranoside
(Structure V) obtained in the proceeding step was taken in dry
pyridine (40 ml) and dry distilled in chloroform (40 ml). To the
reaction mixture, acetic anhydride (13.9 ml) was added. The
solution was mixed gently, sealed and stored at room temperature
for 72 hours. The solution was then diluted with chloroform. The
total organic layer was placed in a separatory funnel and washed
with saturated aqueous sodium bicarbonate solution once, followed
by washing with saturated brine solution. The organic layer was
passed over anhydrous sodium sulfate, followed by evaporation on a
rotary evaporator. The residue was co-evaporated with toluene three
times. The gummy mass was dried using a direct vacuum line for 2
hours. Anhydrous ethanol was added to the gummy mass. The solution
was kept at 4.degree. C. for 2 hours. The solid obtained was
filtered and washed with cold ethanol. The solid was transferred in
a round bottom flask and dried on high vacuum direct line at
37.degree. C. for 12 hours. The processes resulted in a yield of
4.5 grams of an off white product. The product was analyzed by 1H
NMR and Mass spectral analysis.
Referring to FIGS. 3, 5 and 7, the synthesis of modified
phosphoramidites are illustrated and carried out according to
Schemes 4, 6 and 8 respectively and the individual steps outlined
in below. FIGS. 3, 5 and 7 show illustrative examples of
phosphoramidites having nucleobases uracil, cytosine, and adenine.
Phosphoramidites having other nucleobases such as guanine or
modified nucleobases can be synthesized using the same or similar
steps. Accordingly, the following examples are illustrative only
and not meant to be limiting.
Synthesis of 2',3',5'-tri-hydroxy-2',3',4',5',5'' penta deuterium
.beta.-D ribofuranosy Uridine (Structure IX)
A mixture of Uracil (compound VII; 0.5 gm; 4.46 mmole), hexamethyl
disilazane (15 ml) and ammonium sulphate (20 mg 0.15 mmol) was
boiled under reflux until the Uracil was dissolved, approximately
15 hours. Subsequently, hexamethyldisilazane was evaporated under
vacuum & toluene is added. The mixture was shaken and solvents
were evaporated out to obtain a residual solid consisting of
trimethyl silylated uracil. The solid residue was used without
purification for coupling. Freshly distilled 1,2 dichloro ethane
(freshly distilled over CaH.sub.2), (16 ml) was to the residue. The
mixture was stirred at 40.degree. C., followed by addition of
stannic chloride (1.13 ml; 1.46 mmole) at the 40.degree. C.
temperature. The reaction was continued for 15 minutes at
40.degree. C. Deuterated 1-acetate .alpha./.beta.-D ribofuranoside
(structure VI; 1.81 gm; 3.56 mmol) solution in 1,2 dichloro ethane
(freshly distilled over CaH.sub.2) was placed in a pressure
equalizing funnel and mounted on top of the reaction flask above.
The solution was added drop wise and the reaction was boiled under
reflux for 2.5 hr. The reaction mixture was cooled and stirred in a
saturated sodium bicarbonate solution for 1.5 hr. The reaction
mixture was filtered through a bed of celite powder. The organic
layer was separated and passed through anhydrous sodium sulphate.
The reaction mixture was evaporated under vacuum. And checked TLC
in chloroform: methanol (8:2). A gummy mass obtained was
chromatographed on a column (1.5''.times.14 cm) of silica (70:230
mesh) (100 gm) with EtOAc: Hexane (6:4) as an eluant. Fractions
were monitored by TLC. The R.sub.f value was 0.46 in
chloroform:methanol (8:2). Pure fractions monitored by UV
visualization, combined, concentrated on rotary evaporator and the
compound having the structure VIII was obtained as a foam (yield;
1.7 gm).
A mixture of structure VIII (1.7 gm) in pyridine (20 ml) and
aqueous ammonia solution (37% w/v, 20 ml) was kept in a tightly
sealed flask at 37.degree. C. for 48 hours. The mixture was then
evaporated in vacuum and co-evaporated with isopropyl alcohol to
dryness. A solution of residue in dichloromethane was applied to a
column (2.times.15 cm) packed with Silica Gel (70:230) (100 gm) in
chloroform, followed by chloroform:methanol:85:15 (V:V). The pure
fraction as visualized by UV, and was evaporated to yield a
compound powder of structure IX,
2',3',5'-tri-hydroxy-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl Uridine, (yield; 700 mg; 93.3%), See FIGS. 13A-13C.
Rf; 0.4 system; chloroform: methanol (85:15).UV; maxima at 260
(0.494), Emax; 7826.22.
Synthesis of 5'-O-dimethoxy trityl-2',3',4',5',5'' penta deuterium
.beta.-D ribofuranosyl Uridine (Structure X)
2',3',5'-tri-hydroxy-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl Uridine (structure IX; 0.7 gm; 0.175 mmol) was dried
with dry pyridine two times followed by addition of dry pyridine
(10 ml). The solution was stirred and cooled to 0.degree. C. with a
drying tube attached. To the solution was added 4, 4, dimethoxy
trityl chloride (DMT-Cl; 1.16 gm; 3.42 mmole) in two portions at
one hour intervals. The progress of the reaction was monitored by
TLC in Chloroform (85:15). After completion of reaction (approx. 4
hours), the reaction mixture was quenched with cooled methanol (5
ml), followed by removal of solvent on a rotary evaporator. The
residual gum was taken in chloroform and washed with saturated
bicarbonate solution once, followed by washing with brine solution
once. The crude product obtained after removal of the solvent was
chromatograph on a column of silica Gel (70:230 mesh size) (150 gm)
with chloroform: methanol (95:5) as an eluant. Fractions were
monitored by TLC and visualized by UV. Rf 0.4 in chloroform:
methanol (95:05). Pure fractions were combined and evaporated to
give an almost colorless foam, yield; of 1.3 gm; 86.6%, UVmax at
250 nm; Emax of 11,671. The product was analyzed by one or more of
the following HPLC, UV, 1H NMR, mass spectral data and/or .sup.31P
NMR, see FIGS. 14A-14D.
Synthesis of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl-Uridine XI and
5'-O-dimethoxytrityl-3'-O-tert-Butyldimethylsilyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl-Uridine XII
Compound 5'-O-dimethoxy trityl-2',3',4',5',5'' penta deuterium
.beta.-D ribofuranosyl Uridine (structure IX; 1.3 gm; 2.36 mmole)
was dried by co-evaporation with anhydrous acetonitrile and under
vacuum for several hours. The dried product was added to anhydrous
tetrahydrofuran (THF, 13 ml). To the solution was added silver
nitrate (AgNo3 0.5 gm, 2.94 mmole) under anhydrous condition with a
drying tube on top of the reaction flask. To the mixture was added
dry pyridine (0.69 ml; 8.54 mmole). The reaction mixture was
stirred for 10 minute at room temperature. Subsequently, tert butyl
dimethyl silyl chloride (TBDMS-Chloride, 0.53 gm, 3.52 mmole) was
added under anhydrous conditions. The reaction mixture was sealed
and stirred at room temperature for at 2.5 hours. The progress of
the reaction was monitored by TLC and visualized under UV. The TLC
solvent system was chloroform: Hexane: Acetone (65:25:10). The
crude product showed formation of both the 2' isomer (Structure XI)
and 3' isomer. The comparative analysis on TLC with unmodified 2'
and 3'-isomers was carried out and the spots co-migrated.
After the usual work up, the crude product was chromatographed on a
column of silica gel (230:400 mesh) with a solvent system
consisting of chloroform:Hexane:Acetone: 65:25:10. The fractions
were monitored by TLC and visualized by UV. The R.sub.f value was
0.38 in the same solvent system. Combined pure fractions were
evaporated to give a foam having a yield of 800 mg, of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-2',3',4',5',5''penta
deuterium .beta.-D ribofuranosyl-Uridine 50.9% UV ^ max at 250 nm
(0.350); Emax of 1634. The 3'-isomer
1-(5-O-dimethoxytrityl-3-O-tert-Butyldimethylsilyl-2,3,4,5,5' penta
deuterium .beta.-D ribofuranosyl) Uracil XII was not isolated. The
product was analyzed by one or more of the following HPLC, UV, 1H
NMR, mass spectral data and/or .sup.31P NMR, see FIGS. 15A-15D.
Synthesis of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-N,
N-diisopropyl cyanoethyl phosphoramidite-2',3',4',5',5'' penta
deuterium .beta.-D ribofuranosyl Uridine (Compound Structure
XIII)
From the synthesis of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl Uridine, Structure XI and
5'-O-dimethoxytrityl-3'-O-tert-Butyldimethylsilyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl Uridine Structure XII, the
2'-TBDMSilyl isomer (structure XI; 430 mg) was thoroughly dried
with anhydrous acetonitrile and placed in a round bottom flask.
Anhydrous tetrahydrofuran (4.3 ml) was added and the solution
purged with Argon and replaced with a stopper. To the solution,
under stirring, was added 2,4,6-collidine (430 microliter; 5
equivalents), followed by addition of 1-methyl imidazole (51
microliters; 1.0 equivalents). The solution was stirred at room
temperature and N,N-diisopropylamino cyanoethyl phosphonamidic
chloride (phosphorylating reagent, ChemGenes Catalog No. RN-1505;
290 microliters; 2 equivalents) was quickly added. After 70
minutes, the reaction was complete, and it was worked up by
dilution with chloroform. The organic layer was placed in a
separatory funnel and washed with saturated aqueous sodium
bicarbonate, followed by further washing of the organic layer with
brine solution. The organic layer was passed over anhydrous sodium
sulfate. The solution was concentrated on a rotary evaporator. The
TLC was checked in the system ethyl acetate: hexane: triethylamine
(30:60:10). The crude product was purified on a column of silica
gel (230-400 mesh) column diameter (30 cm.times.1.5 cm). The pure
fractions were monitored by TLC and combined and then concentrated.
A colorless, foamy product was obtained having a dry weight of 300
mg. The product was analyzed by HPLC, UV, 1H NMR, mass spectral
data and 31 P NMR, see FIGS. 16A-16F.
Solid supports attached with deuterium labeled nucleosides are
required for the synthesis of oligonucleotides. Solid support bound
with deuterium labeled nucleosides after oligonucleotide synthesis
result in deuterium labeled nucleoside at the 3'-end of the
oligonucleotide. In this process the oligonucleotide synthesis is
carried out from 3'-end to 5'-end direction (conventional
oligonucleotide synthesis). The instant invention discloses methods
for synthesizing deuterium labeled nucleoside-3'-succinate
nucleosides with controlled deuterium label which can vary from
0.1%-98% deuterium at specific positions of the sugar and
purine/pyrimidine bases. The instant invention discloses a process
which incorporates deuterium containing phosphoramidites and solid
supports, which have varying percent of enrichment of deuterium
with a ratio of deuterium and hydrogen ranging from 20:98.
Structure B illustrates a deuterated solid support structure having
the chemical structure of:
##STR00002##
wherein X represents deuterium or hydrogen, R1 represents a
blocking group, R2 independently represents a blocking group, R3
represents a linking molecule, and R4 represents a solid support
and B represents a nucleobase. As described previously, B may be a
natural base, a modified base, or combinations thereof. Linking
molecules are generally known in the art as small molecules which
function to connect a solid support to functional groups. The
preferred linking molecule is succyl-Icaa, but other linking
molecules known to one of skill in the art may be used. The solid
support is generally used to attach to a first nucleoside. In a
preferred embodiment, the solid support is controlled pore glass
(CPG). However, other supports, such as, but not limited to,
oxalyl-controlled pore glass, macroporous polystyrene (MPPS),
aminopolyethyleneglycol, may be used as well.
FIG. 4 illustrates Scheme 3, synthesis of a deuterated
ribonucleoside coupled to a solid support structure, illustrated
herein as
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-succinyl
Icaa-CPG-2',3',4',5',5'' penta deuterium .beta.-D ribofuranosyl
Uridine.
Synthesis of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-O-succinyl
pyridinium salt-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl Uridine (Compound Structure XIV)
The compound,
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl Uridine (XI; 350 mg) was
placed in dry pyridine 3.5 ml and stirred. To the stirred solution
was added succinic anhydride (158 mg; 1.58 mmol), followed by
addition of 4-dimethyl amino pyridine (20 mg; 0.163 mmol). The
reaction mixture was sealed and kept in a water bath and maintained
at 37.degree. C. for 14 hours. The reaction mixture was checked by
TLC and found to be complete. Subsequently, the reaction mixture
was quenched with cold methanol (200 microliters), followed by
solvent removal on a rotary evaporator. The crude reaction mixture
was placed in chloroform and the organic layer was washed with
saturated brine solution. The organic layer was filtered through
anhydrous sodium sulfate and the chloroform solution was removed
under vacuum. The crude compound was purified by a short column
chromatography using chloroform:methanol (95:5) solvent system. The
pure fractions were combined and evaporated. The foamy product was
dried on high vacuum for 6 hours. The Rf value of the product in
this system was 0.3. The process yielded 120 mg. The product was
analyzed by one or more of the following HPLC, UV, 1H NMR, mass
spectral data and/or .sup.31P NMR, see FIGS. 17E and 17F.
Synthesis of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-succinyl
Icaa-CPG-2,'3,'4,'5,'5''penta deuterium .beta.-D ribofuranosyl
Uridine (Compound Structure XV)
The preceding step nucleoside, 3'-succinate-pyridinium salt,
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-succinyl
pyridinium salt-2',3',4',5',5''penta deuterium .beta.-D
ribofuranosyl Uridine (compound structure XIV; 85 mg) was placed in
a round bottom flask and thoroughly dried with anhydrous
acetonitrile, followed by drying under high vacuum using a direct
line for 6 hours. To the solid was added anhydrous acetonitrile (6
ml), followed by addition of
O-(Benzotriazole-1-Y-L)-N,N,N,N-tetramethyl-uronium-hexafluoro-phosphate,
HBTU; (47 mg; 1.1 equivalents). Diisopropyl ethylamine (39
microliters; 2 equivalents). Was then added. To the solution was
added an amino linker Icaa CPG (long chain alkyl amine controlled
Pore Glass; 500 a particle size; a product of Prime Synthesis Inc.,
Pennsylvania; 1.5 g). The mixture was sealed thoroughly and kept at
37.degree. C. for 12 hours. The CPG was filtered, washed with
acetonitrile, followed by diethyl ether. The CPG was air dried
overnight.
The residual amino group was blocked. The dried CPG was placed in
an Erlenmeyer flask, and CAP A solution (a ChemGenes product,
catalog no. RN-1458 consists of acetic anhydride: pyridine:
tetrahydrofuron (10:10:80) 10 ml) was added. The suspension was
kept at room temperature well sealed for 2 hours. The CPG was
filtered, washed with isopropanol, followed by washing with diethyl
ether. The completion of complete blocking of the residual amino
function was checked by ninhydrin test. A negative ninhydrin test
indicates complete capping of residual amino functional group.
Trityl determination of the loaded CPG was carried out. The trityl
value was 44 .mu.mol/g.
Referring to FIG. 5, Scheme 4 illustrates an example of synthesis
of an alternative embodiment of a phosphoramidite in accordance
with the instant invention and having with the nucleobase cytosine
having the structure XXII. The details of the individual steps
involved in the synthesis are outlined below.
Synthesis of 2',3',5'-tri O-benzoyl-2',3',4',5',5'' penta deuterium
.beta.-D ribofuranosyl N.sup.4 benzoyl Cytidine: Structure
XVII)
A mixture of N.sup.4 bz-cytosine; (compound structure XVI; 750 mg;
3.47 mmol), hexamethyl disilazane (HMDS; 19 ml) and ammonium
sulphate (32 mg; 0.24 mmol) was boiled under reflux until the
N.sup.4 bz-cytosine dissolved, approximately 15 hours.
Hexamethyldisilazane was then evaporated under vacuum and toluene
added. The mixture was shaken and the solvents evaporated out to
obtain a residual solid consisting of trimethyl silylated N.sup.4
bz-cytosine. The solid residue was used without purification for
coupling. Freshly distilled 1,2 dichloro ethane (freshly distilled
over CaH.sub.2), 16 ml was added to the residue. The mixture was
stirred at 40.degree. C. Stannic chloride (0.86 ml; 3.29 mmole) was
then added at the 40.degree. C. temperature. The reaction was
continued for 15 minutes at the 40.degree. C. temperature.
Deuterated .beta.-D ribose-1-acetate (structure VI (1.42 gm; 2.79
mmole) solution in 1,2 dichloro ethane (4.3 ml; freshly distilled
over CaH.sub.2) was placed in a pressure equalizing funnel and
mounted on top of the reaction flask. The solution was added drop
wise and the reaction was boiled under reflux for 2.5 hours. The
reaction mixture was cooled and stirred in saturated sodium
bicarbonate solution for 1.5 hours. The reaction was filtered
through a bed of celite powder. The organic layer was separated and
passed through anhydrous sodium sulphate. The reaction mixture was
evaporated under vacuum and checked using TLC, in chloroform:
methanol: (98:02). The R.sub.f value was 0.46. The reaction yielded
2.14 grams.
Synthesis of 2',3',5'-tri Hydroxy-2',3',4',5',5'' penta deuterium
.beta.-D ribofuranosyl N.sup.4 benzoyl Cytidine (Compound Having
Structure XVIII)
A mixture of 2',3',5'-tri O-benzoyl-2',3',4',5',5'' penta deuterium
.beta.-D ribofuranosyl N.sup.4 benzoyl Cytidine, structure XVII,
(2.14 g; 3.22 mmol) in pyridine (20.5 ml) as stirred until
dissolved. Methanol (5 ml) was then added. The solution was cooled
to 0.degree. C. and 2N aqueous sodium hydroxide solution (6.26 ml)
for selective hydrolysis of O-benzoyl groups was added. The
hydrolysis reaction was carried out for 20 minutes at 0.degree. C.
while stirring continued. The reaction mixture was carefully
neutralized to a pH 7.5 with 2N aqueous HCl (7 ml). The solution
was evaporated after addition of pyridine (10 ml). The residue was
co-evaporated with isopropyl alcohol to dryness. The residue was
titrated with distilled water to give a colorless solid. The solid
was filtered, washed with diethyl ether and dried under high
vacuum. A compound having structure XII was obtained as a powder
(yield 1.0 g; 88.49%). The Rf value was 0.5 in chloroform:
methanol:(85:15); UV max. at 260 (0.903), and Emax of 16,000. The
product was analyzed by one or more of the following HPLC, UV, 1H
NMR, mass spectral data and/or .sup.31P NMR, see FIGS. 18A-18D.
Synthesis of 5'-O-dimethoxytrityl-2',3',4',5',5'' penta deuterium
.beta.-D ribofuranosyl N.sup.4 Benzoyl Cytidine (Compound Having
Structure XIX)
Compound 2',3',4',5', 5'' penta deuterium .beta.-D ribofuranosyl
N.sup.4 Benzoyl Cytidine (structure XVIII 1.0 g; 2.88 mmol) was
dried with dry pyridine two times followed by addition of dry
pyridine (10 ml). The solution was stirred and cooled to 0.degree.
C. with a drying tube attached. 4,4, dimethoxy trityl chloride
(DMT-Cl; 1.15 gm; 3.39 mmol) was added to the solution in two
portions at one hour intervals. The progress of the reaction was
monitored by TLC in Chloroform: 85:15. After completion of reaction
(approx. 4 hours), the reaction mixture was quenched with cooled
methanol (5 ml), followed by removal of the solvent on a rotary
evaporator. The residual gum was placed in chloroform and washed
with a saturated bicarbonate solution once, followed by washing
with brine solution. The crude product obtained after removal of
the solvent was chromatographed on a column of silica Gel (70:230
mesh size) (150 gm) with chloroform:Hexane:Acetone (50:30:20).
Fractions were monitored by TLC and visualized by UV. Rf 0.4 in
chloroform:methanol: 94:06. Pure fractions were combined and
evaporated to give almost a colorless foam, (yield; 1.5 gm;
81.08%), UV lambda max at 260 nm; Emax; 16609.66 (260 nm). The
product was analyzed by one or more of the following HPLC, UV, 1H
NMR, mass spectral data and/or .sup.31P NMR, see FIGS. 19A-19D.
Synthesis of 5'-O-dimetoxytrityl-2'-O-terbutyldimethyl
Silyl-2',3',4',5',5'' penta deuterium .beta.-D ribofuranosyl
N.sup.4 benzoyl Cytidine &
5'-O-dimetoxytrityl-3'-O-terbutyldimethyl Silyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl N.sup.4 benzoyl Cytidine
(Compound Having Structures XX & XXI)
Compound 5'-O-dimetoxytrityl-2',3',4',5',5'' penta deuterium
.beta.-D ribofuranosyl N.sup.4 Benzoyl Cytidine (compound XIII; 1.5
gm; 1.95 mmol) was dried by co-evaporation with anhydrous
acetonitrile and under vacuum for several hours. The dried product
was placed in anhydrous tetrahydrofuran (THF; 15 ml). To the
solution was added silver nitrate (AgNo3 0.49 gm; 2.94 mmol) under
anhydrous condition with a drying tube on top of the reaction
flask. Dry pyridine (0.60 ml; 7.26 mmol) was added to the mixture
and stirred for 10 minute at room temperature. Subsequently,
tert-butyldimethyl silyl chloride (TBDMS-Chloride; 0.52 g; 3.52
mmol) under anhydrous conditions to seal the reaction mixture. The
mixture was stirred for 2.5 hours at room temperature. The progress
of the reaction was monitored by TLC and visualized under UV. The
TLC solvent system used first checked using chloroform:
Hexane:Acetone (65:25:10) (R.sub.f value was 0.38) and then using
ethyl acetate:hexane (50:50). The crude product showed formation of
both the 2' isomer (Structure XX) and 3' isomer (Structure XXI).
The comparative analysis on TLC with unmodified 2' and 3'-isomers
was carried out and the spots co-migrated.
The crude product was chromatographed on a column of silica gel
(230:400 mesh) with a solvent system consisting of
chloroform:Hexane:Acetone (65:25:10). The fractions were monitored
by TLC and visualized by UV. The R.sub.f was 0.38 in the same
solvent system. Combined pure fractions were evaporated to give a
foam with a yield of 800 mg; of
5'-O-dimetoxytrityl-2'-O-terbutyldimethyl Silyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl N.sup.4 benzoyl Cytidine
50.9%. UV ^ max at 250 nm (0.350); Emax of 1634. The 3'-isomer,
5'-O-dimetoxytrityl-3'-O-terbutyldimethyl Silyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl) N.sup.4 benzoyl Cytidine
(structure XXI) was not isolated.
Synthesis of 5'-O-dimetoxytrityl-2'-O-terbutyldimethyl
Silyl-3'-N,N-diisopropyl cyanoethyl phosphoramidite-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl) N.sup.4 benzoyl Cytidine
(Compound Having Structure XXII)
From the preceding step, the 2'-TBDMSilyl isomer (compound XX; 430
mg) was thoroughly dried with anhydrous acetonitrile and placed in
a round bottom flask. Anhydrous tetrahydrofuran (2.0 ml) was added
and the solution was purged with Argon and replaced with a stopper.
To the solution under stirring, 2,4,6-collidine (176 microliter; 5
equivalents) was added, followed by addition of 1-methyl imidazole
(21 microliters; 1.0 equivalents). To the stirred solution at room
temperature, N,N-diisopropylamino cyanoethyl phosphonamidic
chloride (phosphorylating reagent, ChemGenes Catalog No. RN-1505;
119 microliters; 2 equivalents) was added. After 75 minutes, the
reaction was found to be complete, and it was worked up by dilution
with chloroform. The organic layer was placed in a separatory
funnel and washed with saturated aqueous sodium bicarbonate,
followed by further washing of the organic layer with brine
solution. The organic layer was passed over anhydrous sodium
sulfate. The solution was concentrated on a rotary evaporator and
checked using TLC with a solvent system of ethyl
acetate:hexane:triethylamine: 50:40:10 and ethyl
acetate:hexane:triethylamine (30:60:10) and (50:40:10).
The crude product was purified on a column of silica gel (230-400
mesh) having a column diameter 30 cm.times.1.5 cm. The column was
run first in the system using ethyl acetate:hexane:triethylamine
(30:60:10) and after removal of upper impurities, the system was
changed to ethyl acetate: hexane: triethylamine (50:40:10). The
pure fractions were, monitored by TLC, were combined and
concentrated. Colorless foamy product was obtained having a dry
weight of 125 mg. The product was analyzed by HPLC, UV, 1H NMR,
Mass spectral data and 31 P NMR, see FIGS. 20A-20F.
Referring to FIG. 6, Scheme 5 illustrates an example of synthesis
of an alternative embodiment of a deuterated solid support
structure, illustrated herein as
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-succinyl
Icaa-CPG-2',3',4',5',5'' penta deuterium .beta.-D ribofuranosyl
N.sup.4 benzoyl Cytidine.
Synthesis of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-O-succinyl
pyridinium salt-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl N.sup.4 benzoyl Cytidine (Compound Having Structure
XXIII)
The compound,
5'-O-dimethoxytrityl-2'-O-tert-butyldimethylsilyl-2',3',4',5',5''
penta deuterium .beta.-D ribofuranosyl N.sup.4 benzoyl Cytidine
(structure XXIII); (300 mg) was placed in 3.0 ml dry pyridine.
Succinic anhydride (120 mg; 1.99 mmol was added to the stirred
solution, followed by addition of 4-dimethyl amino pyridine (14 mg;
0.115 mmol). The reaction mixture was sealed and kept in a water
bath maintained at 37.degree. C. for 14 hours. The reaction mixture
was checked by TLC and determined to be complete. Subsequently, the
reaction mixture was quenched with cold methanol (180 microliters),
followed by solvent removal on a rotary evaporator. The crude
reaction mixture was placed in chloroform and the organic layer was
washed with saturated brine solution. The organic layer was
filtered through anhydrous sodium sulfate and the chloroform
solution was concentrated under vacuum. The crude compound was
purified by a short column chromatography using chloroform:
methanol (95:5) solvent system. The pure fractions were combined
and evaporated. The foamy product was dried on high vacuum for 6
hours. The R.sub.f value of the product in this system was 0.35.
The process yielded 80 mg. The product was analyzed by one or more
of the following HPLC, UV, 1H NMR, mass spectral data and/or
.sup.31P NMR, see FIGS. 21A-21B.
Synthesis of
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-succinyl
Lcaa-CPG-2',3',4',5',5' penta deuterium .beta.-D ribofuranosyl
N.sup.4 benzoyl Cytidine (Compound Having Structure XXIV)
The preceding step nucleoside, 3'-succinate-pyridinium salt,
5'-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-succinyl
pyridinium salt-2',3',4',5',5'' penta deuterium .beta.-D
ribofuranosyl N.sup.4 benzoyl Cytidine (compound structure XXIII;
40 mg) was placed in a round bottom flask and thoroughly dried with
anhydrous acetonitrile, followed by drying under high vacuum using
a direct line for 6 hours. Anhydrous acetonitrile (6 ml) was added
to the dried material, followed by addition of HBTU; (19.2 mg; 1.1
equivalents), followed by addition of diisopropyl ethylamine (16
microliters; 2 equivalents). To the solution was added an amino
linker Lcaa CPG (long chain alkyl amine controlled Pore Glass; 500
A particle size; a product of Prime Synthesis Inc., Pennsylvania;
680 mg). The mixture was sealed thoroughly and kept at 37.degree.
C. for 12 hours. The CPG was filtered, washed with acetonitrile,
and followed by a diethyl ether wash. The CPG was air dried
overnight.
The residual amino groups were blocked. The dried CPG was placed in
an Erlenmeyer flask, and CAP A solution (a ChemGenes product,
catalog no. RN-1458 consists of acetic anhydride: pyridine:
tetrahydrofuran (10:10:80)10 ml was added. The suspension was
sealed and kept at room temperature for 2 hours. Subsequently, the
CPG was filtered, washed with isopropanol, followed by a diethyl
ether wash. The completion of the complete blocking of the residual
amino function was checked by ninhydrin test. A negative ninhydrin
test indicated complete capping of residual amino functional group.
The trityl value indicated a loading of 30 .mu.mol/g.
Referring to FIG. 7, Scheme 6 shows an example of the synthesis of
an alternative embodiment of a phosphoramidite in accordance with
the instant invention, having the nucleobase adenine, structure
XXVIII. The details of the individual steps involved in the
synthesis are outlined below.
Synthesis of 2',3',5' tri-O-benzoyl-2',3',4',5',5''-penta deuterium
.beta.-D ribofuranosyl N.sup.6 Benzoyl Adenosine (Compound Having
Structure XXVI)
A mixture of N.sup.6 bz-adenine (XXV; 760 mg; 3.18 mmol) was placed
in distilled 1,2-dichloroethane and stirred. Bissily acetamidite
(BSA; 3.116 ml; 15.29 mmol) was added and boiled under reflux until
the N.sup.6 bz-adenine was dissolved (15 hr). Subsequently BSA was
evaporated under a vacuum & toluene was added. The mixture was
shaken and the solvents were evaporated to obtain a residual solid
consisting of silylated N.sup.6 bz-adenine. The solid was used
without purification for coupling. Freshly distilled 1,2 dichloro
ethane (50 ml; freshly distilled over CaH.sub.2), was added to the
residue. The mixture was stirred at 40.degree. C., followed by
addition of stannic chloride (0.55 ml; 0.73 mmol) at this
temperature. The reaction was continued for 15 minutes at
40.degree. C. Deuterated .beta.-D ribose-1-acetate (structure VI
(1.29 g; 2.53 mmol) solution in 1,2 dichloro ethane (4.3 ml;
freshly distilled over CaH.sub.2) was placed in a pressure
equalizing funnel and was mounted on top of the reaction flask
above. The solution was added drop wise and the reaction was boiled
under reflux for 2.5 hours.
The reaction mixture was cooled. Saturated sodium bicarbonate
solution was stirred in for 1.5 hours. The reaction was filtered
through a bed of celite powder. The organic layer was separated and
passed through anhydrous sodium sulphate. The reaction mixture was
evaporated under vacuum and checked using TLC, using
chloroform:ethylacetate: triethylamine (47:47:8). The R.sub.f value
was determined to be 0.53.
The crude product was purified by column chromatography (silica
gel; 230-400 mesh), using a solvent system of chloroform:
ethylacetate:triethylamine (47:47:6) The pure fraction, monitored
by TLC, was combined and concentrated on a rotary evaporator. Pure
foamy product was obtained having a yield of 400 mg.
Synthesis of 2',3',5' tri Hydroxy-2',3',4',5',5''-penta deuterium
.beta.-D ribofuranosy) N6 benzoyl Adenosine (Compound Having
Structure XXVII)
The preceding tribenzoyl compound, 2',3',5'
tri-O-benzoyl-2',3',4',5',5''-penta deuterium .beta.-D
ribofuranosyl N6 Benzoyl Adenosine (XXVI; 400 mg) was placed in
pyridine (4.8 ml) and methanol (1.2 ml). The mixture was stirred to
bring the compound to solution. The solution was then cooled to
0.degree. C. with an ice bucket outside. To the solution, 2N NaOH
(1.16 ml) was added, and the basic reaction mixture was hydrolyzed
to remove O-benzoyl groups, for a period of 20 minutes. To the
reaction mixture was then added 2N HCl (cooled to 0.degree. C.).
The addition was done carefully to neutralize the basic solution to
pH 7.5 (using 1.0 ml 2 N HCl). To the reaction mixture, pyridine (5
ml) was added and the solution was concentrated. Co-evaporation
with pyridine (2.times.5 ml) followed the concentration step. The
residue was purified by crystallization by addition of water. The
solid obtained was filtered and washed with diethyl ether. The
solution was checked using TLC, using a solvent system of
chloroform: methanol (85:15). The vacuum dried product had an
R.sub.f value of 0.4 and with a yield of 200 mg.
Synthesis of 5'-O-dimethoxy trityl-2',3',4',5',5''-pentadeuterium
.beta.-D ribofuranosyl N.sup.6 benzoyl Adenosine (Compound Having
Structure XXVIII)
The compound 2',3',5'-tri Hydroxy-2',3',4',5',5''-penta deuterium
.beta.-D ribofuranosyl N6 benzoyl Adenosine (XXVII; 200 mg) was
dried with dry pyridine two times followed by addition of dry
pyridine (2M) under anhydrous conditions. The solution was stirred
and cooled to 0.degree. C. with a drying tube attached. To the
solution was added 4,4, dimethoxy trityl chloride (DMT-Cl; 0.21 g;
0.619 mmol) in one portion. The progress of the reaction was
monitored by TLC in Chloroform (95:05). After completion of the
reaction (approximately 4 hours), the reaction mixture was quenched
with cooled methanol (2 ml). The solvent was then removed on rotary
evaporator. The residual gum was placed in chloroform and washed
with saturated bicarbonate solution once, followed by a single wash
with brine solution. The crude product obtained after removal of
the solvent was chromatographed on a column of silica Gel (70:230
mesh size) (150 gm) with chloroform: methanol (95:5) as an eluant.
Fractions were monitored by TLC and visualized by UV. The R.sub.f
value was 0.38 in chloroform: methanol (95:05). Pure fractions were
combined and evaporated to give almost colorless foam. The process
yielded 80 mg; UVmax at 250 nm; E.sub.max of 11,671. The product
was analyzed by one or more of the following HPLC, UV, 1H NMR, mass
spectral data and/or .sup.31P NMR, see FIGS. 22A-22C.
Oligonucleotide Synthesis: Using Schemes 1-6 to synthesize the
necessary chemical structures, the instant invention describes an
oligonucleotide synthesis process for the production of deuterated
ribonucleotides. Referring to FIG. 8A, illustrative example of a
deuterated ribo-oligonucleotide having structure C1 is shown,
wherein n represents the number of nucleoside units
(ribose+nucleobase) of the oligonucleotide, thereby defining the
oligonucleotide sequence, B represents natural or modified
nucleobase, and X is deuterium, wherein W could be oxygen (O.sup.-)
or Sulfur (S.sup.-); Y could be oxygen (O.sup.-) C1-C18 alkoxy,
C1-18 alkyl; NHR3 with R3 being C1-C18 alkyl or C1-C4
alkoxy-C1-C6-alkyl; NR3R4 in which R3 is as defined above and R4 is
C1-C18 alkyl, or in which R3 is as defined above and R4 is
C1-C18-alkyl, or in which R3 and R4 form together with the nitrogen
atom carrying them, a 5-6 membered heterocyclic ring which can
additionally contain another hetero atom from the series O, S and
N. Alternatively, the oligonucleotide linkage could contain a
Y-group which be replaced with
X--C--(Y.sub.1Y.sub.2Y.sub.3Y.sub.4)--, represented by Formula
II:
##STR00003## wherein W can be oxygen (O.sup.-) or sulfur (S.sup.-);
Y can be singly or multiply hydrogen, methyl, ethyl; X can be an
electron attracting group, such but not limited to, halogen, such
as fluorine, chlorine, or bromine, CN, NO.sub.2, SO.sub.2, aromatic
groups such as but not limited to phenyl thio, phenyl sulfoxy,
phenylsulfonyl. The phenyl ring groups can be substituted with
halogen, CN, NO.sub.2. It is also possible for
[X--C--(Y.sub.1,Y.sub.2)] in formula II to be replaced by CF, CCl,
or CBr.sub.3.
The number of nucleoside units of the oligonucleotide may be for
example 2-200, preferably less than 100, and most preferably
between 2 and 50. The oligonucleotide unit may have deuterium
levels in the range of 1% to 98% accomplished by dilution with cold
material. For example, the oligonucleotide having 100% duteration
may be serially diluted with cold RNA for final concentrations of
between 0.1% and 98%. As illustrated in FIG. 9A, the
oligonucleotide preferably contains a phosphodiester
internucleotide linkage. FIG. 9B illustrates an alternative
embodiment of the deuterated oligonucleotide illustrated in FIG. 8
having a phosphate backbone variant illustrated as, but not limited
to, phosphorothioate internucleotide linkages. Phosphorothioate
modifications have been shown to be useful for delivering
biologically active oligonucleotides, see Protocols for
Oligonucleotides and Analogs, Editor, Sudhir Agarwal, Humana Press,
Totawa, N J, 1993. Moreover, use of variant backbones such as
phosphorothioate can be useful in resisting degradation by cellular
enzymes, thereby providing a more stable modified
oligonucleotide.
The phosphorylating reagents, N,N-diisopropylamino cyanoethyl
phosphonamidic chloride or 2-cyanoethyl, N,N,N,N-tetraisopropyl
phosphane are readily commercially available and were produced by
ChemGenes Corp (Wilmington, Mass.). High purity dimethoxytriphenyl
chloride (DMT-chloride) was obtained from Esscee Biotech India Pvt.
Ltd. High purity pyridine was obtained from Caledon
Laboratories.
The oligonucleotides listed in Table 1 were synthesized using
3'.fwdarw.5' directed deuterated nucleoside-2'-tertbutyl dimethyl
silyl-3'-cyanoethyl phosphoramidites as well as standard or natural
RNA phosphoramidite chemistry in 1 .quadrature.mole scale. The
syntheses were performed on Expedite 8900 synthesizer using
standard RNA 1 .quadrature.mole cycle.
TABLE-US-00001 TABLE 1 Deuterated/Natural Oligonucleotide sequences
synthesized by conventional synthesis method. SEQ ID NO SEQUENCE
(5' to 3,) SEQ ID NO: 1 CUCUCUCUCUCU SEQ ID NO: 2 CAUUGGUUCAAACAU
SEQ ID NO: 3 AGGUUCAAACAU
Following synthesis of the desired oligonucleotide, the controlled
pore glass (CPG) solid support was transferred to a 2 ml microfuge
tube. Oligonucleotides were cleaved from the CPG and deprotected by
incubation for 30 min at 65.degree. C. in 1 ml of 40% methylamine
solution in water. The supernatant was removed and the CPG was
washed with 1 ml of water. Supernatants were pooled and dried. The
t-butyl-dimethylsilyl protecting group was removed from the RNA
residue by treatment with 250 .mu.l of fresh anhydrous
triethylammonium-trihydrogen fluoride at room temperature in
ultrasonic bath for 2 hours. The oligonucleotide was precipitated
by 1.5 ml of n-butanol. The sample was cooled at -20.degree. C. for
1 hour then centrifuged at 10,000 g for 10 minutes. After the
supernatant was decanted, the pellet was washed with n-butanol one
additional time.
The oligonucleotide was then purified by Ion-Exchange HPLC using a
linear gradient in buffer A=(10.0%, 0.5M TRIS and 10.0% ACN), pH
7.5 and buffer B=1.0 M Lithium Chloride in buffer A. The entire
sample was loaded on a Source 15Q column (1.0 cm.times.25 cm) and
eluted with a linear 5% to 75% acetonitrile gradient over 40
minutes. Samples were monitored at 260 nm and peaks corresponding
to the desired oligonucleotide species were collected, and
precipitated by adding 5.0 volume of (2% LiClO.sub.4, in acetone),
followed by centrifuging at 10,000 g for 10 minutes. The
supernatant was decanted, and the pellet was washed with
ethanol.
General Procedure for 1.0 .mu.mol phosphodiester of oligonucleotide
synthesis is described below. Amidites (solid) used for the
specific sequence of interest were individually placed in a 20 mL
expedite bottle and dissolved in a quantity of dry acetonitrile to
make the solution 0.075M. The bottles were flushed with Argon and
shaken after sealing the screw cap promptly to dissolve the solid
completely. The monomer solution bottles were then screwed in to
the synthesizer. In addition, 1.0 um expedite column with Product
5'-O-DMT-2'-O-tert-Butyldimethylsilyl-Uridine-3'-succinyl lcaa--A
support produced by ChemGenes Corp., Cat # N-6104. Natural RNA base
loaded support was prepared and attached to the synthesizer. Table
2 illustrates the oligonucleotide synthesis scheme using an
automatic DNA/RNA Synthesizer.
TABLE-US-00002 TABLE 2 Oligonucleotide synthesis on an automated
DNA/RNA Synthesizer: Wait # of Time Volume Cycles Reagent (sec)
(.mu.l) Cycle 1 Prewash 2 Synthesis Grade Acetonitrile -- 350 RNA
Protocol Cycle 2a Deblock 2 3% TCA/DCM 60 150 Wash 3 Synthesis
Grade Acetonitrile -- 350 Coupling 1 Ribo-sugar (deuterated)
nucleoside 600 255 amidites (0.075 M concentration) Activator 1
5-Ethylthio Tetrazole (0.35 M) 120 Wash 1 Synthesis Grade
Acetonitrile -- 350 Cap A 1 Acetic anhydride/THF/Pyridine 50 120
Cap B 1 N-Methyl imidazole/THF 100 Wash 1 Synthesis Grade
Acetonitrile -- 350 Oxidize 1 0.02 M Iodine in 25 100
Pyridine/THF/Water Wash 3 Synthesis Grade Acetonitrile -- 350
After completion of the synthesis per summary of the key features
as listed in the Table 2, the controlled pore glass (CPG) solid
support was washed with 3.0 ml diethyl ether and transferred to a 2
ml microfuge tube. Oligonucleotide 1 was cleaved from the CPG and
deprotected by incubation for 30 min at 65.degree. C. in 1 ml of
40% methylamine solution in water. The supernatant was removed and
the CPG was washed with 1 ml of water. The supernatants were pooled
and dried. The t-butyl-dimethylsilyl protecting group was removed
from the RNA residue by treatment with 500 .mu.l of fresh 12.0%
solution of tetraethyl ammonium fluoride in DMSO, at 45.degree. C.
in an ultrasonic bath for 1 hour. Oligonucleotide 1 was
precipitated with 1.5 ml of n-butanol. After precipitation, the
sample was cooled at -20.degree. C. for 1 hour then centrifuged at
10,000 g for 10 minutes. The supernatant was decanted, the pellet
was washed with n-butanol one time. A final wash with 500 .mu.l
ethanol was performed. The sample was centrifuged at 10000 rpm for
5 minutes. Following centrifugation, the supernatant was decanted.
The pellet was dissolved in 1000 .mu.l M.Q water. The optical
density, OD, (Crude desalt) of the sample was measured. The
oligonucleotide was then purified by Ion-Exchange HPLC using a
linear gradient in buffer A (10.0%, 0.5M TRIS and 10.0% ACN), pH
7.5 and buffer B (1.0 M Lithium Chloride in buffer A).
The entire sample was loaded on a Source 15Q column (1.0
cm.times.25 cm) and eluted with a linear 5% to 75% acetonitrile
gradient over 40 minutes. Samples were monitored at 260 nm and
peaks corresponding to the desired oligonucleotide species were
collected, and precipitated by adding 5.0 volume of 2% LiClO.sub.4,
in acetone, followed by centrifugation at 10,000 g for 10 minutes.
The supernatant was decanted, and the pellet was washed with
ethanol.
Oligonucleotide Synthesis Example 1: Oligonucleotide1A:
Oligonucleotide 1A was synthesized to have a sequence according to
SEQ ID NO: 1, rC*rU*rC*rU*rC*rU*rC*rU*rC*rU*rC*rU*, wherein r is a
ribose sugar and * represents deuterated ribose resulting from
using deuterium labeled phosphoramidites in the synthesis process.
Oligonucleotide 1 A was synthesized using 5'.fwdarw.3' approach,
directed with deuterated RNA phosphoramidite chemistry in 1
.quadrature.mol scale. The synthesis was performed on Expedite 8900
synthesizer using standard RNA 1 .quadrature.mol cycle and a
coupling time of the monomers with solid support of 10.0
minutes.
The Amidites used were: (A)
1-(5-O-dimethoxytrityl-2-O-tert-Butyldimethylsilyl-3-N,N-diisopropylcyano-
ethyl phosphoramidite-2,3,4,5 penta deuterium .beta.-D
ribofuranosyl) Uracil, structure XIII; and (B)
1-(5-O-dimetoxytrityl-2-O-terbutyldimethyl Silyl-3-N,N-diisopropyl
cyanoethyl phosphoramidite-2,3,4,5 penta deuterium .beta.-D
ribofuranosyl) N.sup.4 benzoyl Cytosine (compound structure XXII).
The solid support used was
1-(5-O-dimethoxytrityl-2-O-tert-Butyldimethylsilyl-3-succinyl
Icaa-CPG-2,3,4,5penta deuterium .beta.-D ribofuranosyl) Uracil
(compound structure XV). Results of capillary electrophoresis
analysis are illustrated in FIGS. 23A-23C.
Oligonucleotide Synthesis Example 2: Oligonucleotide1B:
Oligonucleotide 1B was synthesized to have a sequence according to
SEQ ID NO: 1, rC**rU**rC**rU**rC**rU**rC**rU**rC**rU**rC**rU**
wherein r is a ribose sugar and ** represents a mixture of
deuterated ribose and natural, unmodified ribose modified resulting
from synthesis using deuterium labeled phosphoramidites and a
mixture with natural unmodified nucleoside phosphoramidite in a
ratio of 25:75. Oligonucleotide 1B has approximately 25% deuterium
label was synthesized using 5'.fwdarw.3' directed RNA
phosphoramidite chemistry in 1 .quadrature.mol scale. The synthesis
were performed on Expedite 8900 synthesizer using standard RNA 1
.quadrature.mol cycle and coupling time of the monomers with solid
support 10.0 minute.
The Amidites used were: (A)
1-(5-O-dimethoxytrityl-2-O-tert-Butyldimethylsilyl-3-N,N-diisopropylcyano-
ethyl phosphoramidite-2,3,4,5 penta deuterium .beta.-D
ribofuranosyl) Uracil (structure XIII); (B)
1-(5-O-dimetoxytrityl-2'-O-terbutyldimethyl
Silyl-3'-N,N-diisopropyl cyanoethyl phosphoramidite-2,3,4,5,5'
penta deuterium .beta.-D ribofuranosyl) N.sup.4 benzoyl Cytidine
(XXII); (C) 1-(5-O-dimetoxytrityl-2'-O-terbutyldimethyl
Silyl-3'-N,N-diisopropyl cyanoethyl phosphoramidite-2,3,4,5,5'
penta deuterium .beta.-D ribofuranosyl) N6 Adenosine. Natural RNA
base for mixing natural RNA base in the sequence, ChemGenes Catalog
product, ANP-5674; and (D)
5'-O-DMT-2'-O-tert-Butyldimethylsilyl-Cytidine
N.sup.bz-3'-N,N-diisopropyl cyanoethyl phosphoramidite, Natural RNA
base, for mixing natural RNA base in the sequence, ChemGenes
Catalog product, ANP-5672. The solid supports used were (A)
1-(5-O-dimethoxytrityl-2'-O-tert-Butyldimethylsilyl-3'-succinyl
Icaa-CPG-2,3,4,5,5'penta deuterium .beta.-D ribofuranosyl) Uracil
(structure XV) and (B)
5'-O-DMT-3'-O-tert-Butyldimethylsilyl-Uridine-2'-succinyl Icaa--A
support produced by ChemGenes Corp., Cat # N-6104. Natural RNA base
loaded support was mixed with the Support A listed above in 25:75
ratio to obtain 1.0 micromole column in order to obtain
oligonucleotide 1B consisting of 3'-terminal U with a natural U and
deuterium modified 3'-terminal U in a ratio of 75:25, for mixed
modified RNA base in the sequence.
Oligonucleotide Synthesis Example 3: Oligonucleotide 1C
Oligonucleotide 1C was synthesized to have a sequence according to
SEQ ID NO: 1, rCrUrCrUrCrUrCrUrCrUrCrU wherein r is a ribose unit
consisting of unmodified natural bases Uridine and Cytidine. The
oligonucleotide was synthesized using 3'.fwdarw.5' directed RNA
phosphoramidite chemistry in 1 micro mole scale. The synthesis were
performed on Expedite 8900 synthesizer using standard RNA 1 micro
mole cycle and coupling time of the monomers with solid support
10.0 minute. The amidites used included (A)
5'-O-DMT-2'-O-tert-Butyldimethylsilyl-Uridine-3'-N,N-diisopropyl
cyanoethyl phosphoramidite, Natural RNA base for natural RNA base
sequence, ChemGenes Catalog product, ANP-5674 and (B)
5'-O-DMT-2'-O-tert-Butyldimethylsilyl-Cytidine
N.sup.bz-3'-N,N-diisopropyl cyanoethyl phosphoramidite, Natural RNA
base for mixing natural RNA base sequence, ChemGenes Catalog
product, ANP-5672. The sold supports used was
5'-O-DMT-3'-O-tert-Butyldimethylsilyl-Uridine-2'-succinyl Icaa--A
support produced by ChemGenes Corp., Cat # N-6104. Natural RNA base
loaded support.
Oligonucleotide Synthesis Example 4: Oligonucleotide 2:
Oligonucleotide 2, was synthesized to have a sequence according to
SEQ ID NO: 2, consisting of unmodified natural bases uridine,
cytidine and adenosine. The oligonucleotide was synthesized using
5'.fwdarw.3' directed RNA phosphoramidite chemistry in 1 micro mole
scale. The synthesis were performed on Expedite 8900 synthesizer
using standard RNA 1 .quadrature.mol cycle and coupling time of the
monomers with solid support 10.0 minute. The amidites used included
(A)
5'-O-DMT-2'-O-tert-Butyldimethylsilyl-Uridine-3'-N,N-diisopropyl
cyanoethyl phosphoramidite, Natural RNA base for natural RNA base
sequence, ChemGenes Catalog product, ANP-5674; (B)
5'-O-DMT-2'-O-tert-Butyldimethylsilyl-Cytidine
N.sup.bz-3'-N,N-diisopropyl cyanoethyl phosphoramidite, Natural RNA
base for natural RNA base sequence, ChemGenes Catalog product,
ANP-5672; (C) 5'-O-DMT-2'-O-tert-Butyldimethylsilyl-Adenosine
N.sup.bz-3'-N,N-diisopropyl cyanoethyl phosphoramidite, Natural RNA
base for natural RNA base sequence, ChemGenes Catalog product,
ANP-5671; (D) 5'-O-DMT-2'-O-tert-Butyldimethylsilyl-Guanosine
N.sup.ibu-3'-N,N-diisopropyl cyanoethyl phosphoramidite, Natural
RNA base for natural RNA base sequence, ChemGenes Catalog product,
ANP-5673. The solid support used included
5'-O-DMT-3'-O-tert-Butyldimethylsilyl-Uridine-2'-succinyl Icaa--A
support produced by ChemGenes Corp., Cat # N-6104. Natural RNA base
loaded support. Results of capillary electrophoresis analysis are
illustrated in FIGS. 24A-24C.
Oligonucleotide Synthesis Example 5: Oligonucleotide 3:
Oligonucleotide 3 was synthesized to have a sequence of SEQ ID NO:
3, consisting of unmodified natural bases Uridine and cytidine,
guanidine and adenosine. The oligonucleotide was synthesized using
5'.fwdarw.3' directed RNA phosphoramidite chemistry in 1 micro mole
scale. The synthesis were performed on Expedite 8900 synthesizer
using standard RNA 1 micro mole cycle and coupling time of the
monomers with solid support 10.0 minute. The Amidites used included
(A)
5'-O-DMT-2'-O-tert-Butyldimethylsilyl-Uridine-3'-N,N-diisopropyl
cyanoethyl phosphoramidite, Natural RNA base for natural RNA base
sequence, ChemGenes Catalog product, ANP-5674; (B)
5'-O-DMT-2'-O-tert-Butyldimethylsilyl-Cytidine
N.sup.bz-3'-N,N-diisopropyl cyanoethyl phosphoramidite, Natural RNA
base for natural RNA base sequence, ChemGenes Catalog product,
ANP-5672; (C) 5'-O-DMT-2'-O-tert-Butyldimethylsilyl-Adenosine
N.sup.bz-3'-N,N-diisopropyl cyanoethyl phosphoramidite, Natural RNA
base for natural RNA base sequence, ChemGenes Catalog product,
ANP-5671, and (D) 5'-O-DMT-2'-O-tert-Butyldimethylsilyl-guanosine
N.sup.ibu-3'-N,N-diisopropyl cyanoethyl phosphoramidite, Natural
RNA base for natural RNA base sequence, ChemGenes Catalog product,
ANP-5673. The solid support used was
5'-O-DMT-3'-O-tert-Butyldimethylsilyl-Uridine-2'-succinyl lcaa--A
support produced by ChemGenes Corp., Cat # N-6104. Natural RNA base
loaded support. Results of capillary electrophoresis analysis are
illustrated in FIGS. 25A-25C.
Several preferred RNA sequences having sugar labeled with deuterium
will be synthesized and used for biological assays and testing
according to the methodology described above, see Table 2. The
steps involved in the synthesis are not expected to cause loss of
any deuterium and the deuterium/hydrogen ratio is expected to be
maintained.
Table 2. Additional Deuterated/Natural Oligonucleotide sequences to
be synthesized by conventional synthesis method.
TABLE-US-00003 SEQ ID SEQUENCE NUMBER (5' to 3' prime) SEQ ID NO. 4
CAUUGGUUCAAACAU SEQ ID NO. 5 UUGAUGAAACAU SEQ ID NO. 6 CAGUUCAAACAU
SEQ ID NO. 7 GACCAGUUCAAACAU SEQ ID NO. 8 AGGUUCAAACAU SEQ ID NO. 9
AAACGCCUCCAU SEQ ID NO. 10 AAAUGAAAAUGUCAU SEQ ID NO. 11
AAAUUCUAACAU SEQ ID NO. 12 UUCAAAUUCUAACAU
Oligonucleotide Synthesis Example 6: Oligonucleotide 4: Using the
procedures outlined above, Oligonucleotide 4 having SEQ ID NO: 4,
having a sequence of r-C*A*U*U*G*G*U*U*C*A*A*A*C*A*U* where r is a
ribo-oligonucleotide or an RNA sequence; * denotes a partially of
fully deuterated ribose, such as 2,3,4,5,5' pentadeuterium-D
ribofuranoside attached to each nucleoside unit of the RNA molecule
with a natural phosphodiester backbone, as illustrated in FIG. 8,
can be synthesized. Oligonucleotide 4 having SEQ ID NO: 4 can also
be synthesized to have a sequence of
r-C*p(s)A*p(s)U*p(s)U*p(s)G*p(s)G*p(s)U*p(s)U*p(s)C*p(s)A*p(s)A*p(s)A*p(s-
)C*p(s)A*p(s)U* where r is a ribo-oligonucleotide or an RNA
sequence; ** denotes a partially or fully deuterated ribose, such
as 2,3,4,5,5' pentadeuterium-D ribofuranoside attached to each
nucleoside unit of the RNA molecule, p(s) denotes internucleotide
phosphorothioate. Additionally, Oligonucleotide 4 having SEQ ID NO:
4 may be synthesized, using deuterated phosphoramidites and natural
phosphoramidites, to consist of a mixture of partially or fully
deuterated ribose and natural ribose attached to the nucleobases
with a natural phosphodiester linkages, or variant nucleotide
linkages such as a phosphorothioate linkage. As used herein, the
term partially refers to one or more positions on the sugar and/or
base portion that does not include a deuterium. Additionally, the
term could refer to synthesized oligonucleotides that include a mix
of ribose units that are deuterated and ribose units that are not
deuterated as part of the backbone. Results of capillary
electrophoresis analysis are illustrated in FIGS. 26A-26C.
Oligonucleotide Synthesis Example 7: Oligonucleotide 5: Using the
procedures outlined above, Oligonucleotide 7 having SEQ ID NO: 5,
having a sequence of r-U*U*G*A*U*G*A*A*A*C*A*U* where r is a
ribo-oligonucleotide or an RNA sequence; * denotes a partially or
fully deuterated ribose, such as 2,3,4,5,5'-pentadeuterium-D
ribofuranoside attached to each nucleoside unit of the RNA molecule
with a natural phosphodiester backbone, as illustrated in FIG. 8,
can be synthesized. Oligonucleotide 7 having SEQ ID NO:5 can also
be synthesized to have a sequence of
r-U*p(s)U*p(s)G*p(s)A*p(s)U*p(s)G*p(s)A*p(s)A*p(s)A*p(s)C*p(s)A*p(s)U*
where r is a ribo-oligonucleotide or an RNA sequence; ** denotes a
partially or fully deuterated ribose, such as 2,3,4,5,5'
pentadeuterium-D ribofuranoside attached to each nucleoside unit of
the RNA molecule, p(s) denotes internucleotide phosphorothioate.
Additionally, Oligonucleotide 7 having SEQ ID NO: 5 may be
synthesized, using deuterated phosphoramidites and nature
phosphoramidites, having a mixture of partially or fully deuterated
ribose and natural ribose attached to each nucleobase and having a
natural phosphodiester nucleotide linkage, or variant linkages such
as phosphorothioate linkage. Results of capillary electrophoresis
analysis are illustrated in FIGS. 25A-25C.
Oligonucleotide Synthesis Example 8: Oligonucleotide 6: Using the
procedures outlined above, Oligonucleotide 6 having SEQ ID NO: 6
having a sequence of r-C*A*G*U*U*C*A*A*A*C*A*U* where r is a
ribo-oligonucleotide or an RNA sequence; * denotes a partially or
fully deuterated ribose, such as 2,3,4,5,5' pentadeuterium-D
ribofuranoside attached to each nucleoside unit of the RNA molecule
with a natural phosphodiester backbone, as illustrated in FIG. 8,
can be synthesized. Oligonucleotide 6 having SEQ ID NO: 6 can also
be synthesized to have a sequence of
r-C*p(s)A*p(s)G*p(s)U*p(s)U*p(s)C*p(s)A*p(s)A*p(s)A*p(s)C*p(s)A*p(s)U*
where r is a ribo-oligonucleotide or an RNA sequence; wherein *
denotes a partially or fully deuterated ribose, such as 2,3,4,5,5'
pentadeuterium-D ribofuranoside attached to each nucleoside unit of
the RNA molecule, p(s) denotes internucleotide phosphorothioate.
Additionally, Oligonucleotide 6 having SEQ ID NO: 6 may be
synthesized, using deuterated phosphoramidites and natural
phosphoramidites, having a mixture of partially or fully deuterated
ribose and natural ribose attached to the nucleobases with a
natural phosphodiester nucleotide linkage, or a variant linkage
such as phosphorothioate linkage.
Oligonucleotide Synthesis Example 9: Oligonucleotide 7: Using the
procedures outlined above, Oligonucleotide 7, having SEQ ID NO: 7
having a sequence of r-G*A*C*C*A*G*U*U*C*A*A*A*C*A*U* where r is a
ribo-oligonucleotide or an RNA sequence; * denotes a partially or
fully deuterated ribose, such as 2,3,4,5,5' pentadeuterium-D
ribofuranoside attached to each nucleoside unit of the RNA molecule
with a natural phosphodiester backbone, as illustrated in FIG. 8,
was synthesized. Oligonucleotide 7, having SEQ ID NO: 7 can also be
synthesized to have a sequence of
r-C*p(s)A*p(s)G*p(s)U*p(s)U*p(s)C*p(s)A*p(s)A*p(s)A*p(s)C*p(s)A*p(s)U*,
wherein * where r is a ribo-oligonucleotide or an RNA sequence; **
denotes a partially of fully deuterated ribose, such as 2,3,4,5,5'
pentadeuterium-D ribofuranoside attached to each nucleoside unit of
the RNA molecule, p(s) denotes internucleotide phosphorothioate.
Additionally, Oligonucleotide 7, having SEQ ID NO: 7 may be
synthesized, using deuterated phosphoramidites and nature
phosphoramidites, having a mixture of partially or fully deuterated
ribose and natural ribose attached to the nucleobases, and a
natural phosphodiester nucleotide linkage, or variant linkages such
as phosphorothioate linkage.
Oligonucleotide Synthesis Example 10: Oligonucleotide 8: Using the
procedures outlined above, Oligonucleotide 8 having SEQ ID NO:8
having a sequence of r-A*G*G*U*U*C*A*A*A*C*A*U* where r is a
ribo-oligonucleotide or an RNA sequence; * denotes a partially or
fully deuterated ribose, such as 2,3,4,5,5' pentadeuterium-D
ribofuranoside attached to each nucleoside unit of the RNA molecule
with a natural phosphodiester backbone, as illustrated in FIG. 8,
can be synthesized. Oligonucleotide 8 having SEQ ID NO:8 can also
be synthesized to have a sequence of
r-A*p(s)G*p(s)G*p(s)U*p(s)U*p(s)C*p(s)A*p(s)A*p(s)A*p(s)C*p(s)A*p(s)U*,
wherein * where r is a ribo-oligonucleotide or an RNA sequence; **
denotes a partially or fully deuterated ribose, such as 2,3,4,5,5'
pentadeuterium-D ribofuranoside attached to each nucleoside unit of
the RNA molecule, p(s) denotes internucleotide phosphorothioate.
Additionally, Oligonucleotide 8 having SEQ ID NO:8 may be
synthesized, using deuterated phosphoramidites and nature
phosphoramidites, having a mixture of partially or fully deuterated
ribose and natural ribose attached to the nucleobases, and a
natural phosphodiester linkage, or variant nucleotide linkage, such
as a phosphorothioate linkage.
Oligonucleotide Synthesis Example 11: Oligonucleotide 9: Using the
procedures outlined above, Oligonucleotide 11 having SEQ ID NO: 11
having a sequence of r-A* A* A* C* G* C* C* U* C* C* A* U* where r
is a ribo-oligonucleotide or an RNA sequence; * denotes a partially
or fully deuterated ribose, such as 2,3,4,5,5' pentadeuterium-D
ribofuranoside attached to each nucleoside unit of the RNA molecule
with a natural phosphodiester backbone, as illustrated in FIG. 8,
was synthesized. Oligonucleotide 11 having SEQ ID NO: 11 can also
be synthesized to have a sequence of r-A*p(s) A*p(s) A*p(s) C*p(s)
G*p(s) C*p(s) C*p(s)U*p(s)C*p(s)C*p(s)A*p(s)U*, wherein * where r
is a ribo-oligonucleotide or an RNA sequence; ** denotes a
partially or fully deuterated ribose, such as 2,3,4,5,5'
pentadeuterium-D ribofuranoside attached to each nucleoside unit of
the RNA molecule, p(s) denotes internucleotide phosphorothioate.
Additionally, Oligonucleotide 11 having SEQ ID NO: 11 may be
synthesized, using deuterated phosphoramidites and nature
phosphoramidites, having a mixture of partially or fully deuterated
ribose and natural ribose attached to nucleobases, and a natural
phosphodiester linkage, or variant nucleotide linkage, such as a
phosphorothioate linkage.
Oligonucleotide Synthesis Example 12: Oligonucleotide 10: Using the
procedures outlined above, Oligonucleotide 10 having SEQ ID NO: 10,
having a sequence of r-A*A*A*C*G*C*C*U*C*C*A*U* where r is a
ribo-oligonucleotide or an RNA sequence; * denotes a partially or
fully deuterated ribose, such as 2,3,4,5,5' pentadeuterium-D
ribofuranoside attached to each nucleoside unit of the RNA molecule
with a natural phosphodiester backbone, as illustrated in FIG. 8,
can be synthesized. Oligonucleotide 10 having SEQ ID NO: 10 can
also be synthesized to have a sequence of
r-rA*p(s)A*p(s)A*p(s)U*p(s)G*p(s)A*p(s)A*p(s)A*p(s)A*p(s)U*p(s)*G*p(s)*U*-
p(s)*p(s)Ap(s)U**, wherein * where r is a ribo-oligonucleotide or
an RNA sequence; ** denotes a partially or fully deuterated ribose,
such as 2,3,4,5,5' pentadeuterium-D ribofuranoside attached to each
nucleoside unit of the RNA molecule, p(s) denotes internucleotide
phosphorothioate. Additionally, Oligonucleotide 10 having SEQ ID
NO: 10 may be synthesized, using deuterated phosphoramidites and
nature phosphoramidites, having a mixture of partially or fully
deuterated ribose and natural ribose attached to a nucleobase, and
a natural phosphodiester linkage, or variant nucleotide linkage,
such as a phosphorothioate linkage.
Oligonucleotide Synthesis Example 13: Oligonucleotide 11: Using the
procedures outlined above, Oligonucleotide 11 having SEQ ID NO: 11,
having a sequence of r-A*A*A*U*U*C*U*A*A*C*A*U*, where r is a
ribo-oligonucleotide or an RNA sequence; * denotes a partially or
fully deuterated ribose, such as 2,3,4,5,5' pentadeuterium-D
ribofuranoside attached to each nucleoside unit of the RNA molecule
with a natural phosphodiester backbone, as illustrated in FIG. 8,
was synthesized. Oligonucleotide 11 having SEQ ID NO: 11 can also
be synthesized to have a sequence of
r-A*p(s)A*p(s)A*p(s)U*p(s)U*p(s)C*p(s)U*p(s)A*p(s)A*p(s)C*p(s)A*p(s)U*,
wherein * where r is a ribo-oligonucleotide or an RNA sequence; **
denotes a partially of fully deuterated ribose, such as 2,3,4,5,5'
pentadeuterium-D ribofuranoside attached to each nucleoside unit of
the RNA molecule, p(s) denotes internucleotide phosphorothioate.
Additionally, Oligonucleotide 11 having SEQ ID NO: 11 may be
synthesized, using deuterated phosphoramidites and nature
phosphoramidites, having a mixture of partially or fully deuterated
ribose and natural ribose attached to nucleobases, and a natural
phosphodiester linkage, or variant nucleotide linkages such as a
phosphorothioate linkage.
Oligonucleotide Synthesis Example 14: Oligonucleotide 12 Using the
procedures outlined above, Oligonucleotide 12 having SEQ ID NO: 12,
having a sequence of r-U*U*C*A*A*A*U*U*C*U*A*A*C*A*U*, wherein r is
a ribo-oligonucleotide or an RNA sequence; * denotes a partially or
fully deuterated ribose, such as 2,3,4,5,5' pentadeuterium-D
ribofuranoside attached to each nucleoside unit of the RNA molecule
with a natural phosphodiester backbone, as illustrated in FIG. 8,
can be synthesized. Oligonucleotide 12 having SEQ ID NO: 12 can
also be synthesized to have a sequence of
r-U*p(s)U*p(s)C*p(s)A*p(s)A*p(s)A*p(s)U*p(s)U*p(s)C*p(s)U*p(s)A*p(s)A*p(s-
)C*p(s)A*p(s)U*, wherein * where r is a ribo-oligonucleotide or an
RNA sequence; ** denotes a partially or fully deuterated ribose,
such as 2,3,4,5,5' pentadeuterium-D ribofuranoside attached to each
nucleoside unit of the RNA molecule, p(s) denotes internucleotide
phosphorothioate. Additionally, Oligonucleotide 12 having SEQ ID
NO: 12 may be synthesized, using deuterated phosphoramidites and
nature phosphoramidites, having a mixture of partially or fully
deuterated ribose and natural ribose attached to nucleobases, and a
natural phosphodiester linkage, or variant nucleotide linkage such
as a phosphorothioate linkage.
The deuterated oligonucleotide illustrated in FIG. 8 can further be
modified to provide additional oligonucleotides, see FIGS. 9C-9I.
As illustrated in FIG. 9C, the general structure has been modified
so that at least one bond between nuclosides includes a 3 prime to
3 prime linkage. A shown in the figure, the phosphate group (OWPYO)
interlinks the third ribose sugar to the last ribose sugar. In this
case, the phosphate group bonded to the 3 prime carbon of third
ribose sugar unit interlinks to the last ribose sugar unit by
bonding to the 3 prime carbon. While the modified oligonucleotide
is shown having a single 3 prime to 3 linkage between the third
ribose and the last ribose, the oligonucleotide may contain 2 or
more 3 prime to 3 prime linkages and/or the 3 prime to 3 prime
linkage can be between any two ribose units.
The 3' to 3' linkage oligonucleotide synthesis is expected to be
carried out using synthesis schemes described previously, with
modifications. In order to synthesize oligonucleotides illustrated
in FIG. 9C, synthesis of 3'-DMT-5'-hydroxy-2'-TBDMS nucleosides.
See FIG. 9J may be carried out. Subsequently, synthesis of
5'-succinyl linker--solid support-3'-DMT-2'-TBDMS nucleoside
phosphoramidites, see FIG. 9K, may be carried out by known
procedures, such as the procedure described in Scheme 7, FIG. 9L.
Dueterated oligonucleotide analogs with terminal 3', 3'
internucleotide linkages is expected to provide stability to
nucleases and reduced cleavage of such oligonucleotides in human
serum. Incorporation of such terminal 3', 3' is expected to enhance
the biological function of deuterated oligonucleotides.
FIG. 9D illustrates the deuterated oligonucleotide illustrated in
FIG. 8 having an additional phosphate group linkage. The modified
oligonucleotide comprises at least one bond, or linkage between
nucleosides having a 5 prime to 5 prime linkage. As shown in the
figure, the phosphate group (OWPYO) interlinks the first ribose
sugar to the second ribose sugar. In this case, the phosphate group
bonded to the 5 prime carbon of first ribose sugar unit interlinks
to the second ribose sugar unit by bonding to the 5 prime carbon.
While the modified oligonucleotide is shown having a single 5 prime
to 5 linkage between the third ribose and the last ribose, the
oligonucleotide may contain 2 or mores prime to 5 prime linkages
and/or the 5 prime to 5 prime linkage can be between any two ribose
units.
The oligonucleotide synthesis is expected to be carried out from
3'-5' direction as described previously. The nucleoside
phosphoramidite, see structure 9M-1 illustrated in FIG. 9M will be
used to allow synthesis of oligonucleotides of illustrated in FIG.
9D. The phosphoramidites of formula III-C will be synthesized from
the nucleosides, 3'-DMT-deuterated nucleosides, see FIG. 9J.
Dueterated oligonucleotide analogs with terminal 5', 5'
internucleotide linkages is expected to provide stability to
nucleases and reduced cleavage of such oligonucleotides in human
serum. Incorporation of such terminal 3', 3' is expected to enhance
the biological function of deuterated oligonucleotides.
FIG. 9E illustrates the deuterated oligonucleotide illustrated in
FIG. 8 to include a detection molecule, label or tag. Deuterated
oligonucleotides carrying detection molecules, labels or tags can
be used to obtain information relating to, for example,
intracellular distribution of fluorescence emitting
oligonucleotides. Deuterated oligonucleotides bearing detection
molecules can be valuable for learning of transport of, cellular
uptake in cytoplasm and nuclei as well as for preferential
accumulation of such oligonucleotides. It is further anticipated
that deuterated oligonucleotides may be useful in determining
cytoplasmic location and measurement of inhibitory activity, and
the development of more efficient oligonucleotides for import
inside a cell. As illustrated in FIG. 9E, the modified
oligonucleotide includes detecting molecule X. The detecting
molecule X is shown bonded to the base B of the first nucleoside.
Preferably, the detecting molecule attaches to the base B via a
linker molecule. Such placement is illustrative only and X can be
bonded to any base B and at any position on the base. In addition,
the modified oligonucleotide can include multiple detecting
molecules or tags, X1, X2, X3, see FIG. 9F. Detecting molecules X1,
X2, X3 may be the same molecule or independent, different
molecules. Preferably, the detecting molecule X1, X2, X3 is a
flurophore or chromophore. Suitable fluorophores include, but are
not limited to, xanthene dyes, such as fluorescein or rhodamine
dyes, including 6-carboxyfluorescein (FAM),
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE),
tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G),
N,N,N;N'-tetramethyl-6-carboxyrhodamine (TAMRA),
6-carboxy-X-rhodamine (ROX). Suitable fluorophores also include the
naphthylamine dyes that have an amino group in the alpha or beta
position. For example, naphthylamino compounds include
1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene
sulfonate and 2-p-toluidinyl-6-naphthalene sulfonate,
5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Other
fluorophores include coumarins, such as
3-phenyl-7-isocyanatocoumarin; acridines, such as
9-isothiocyanatoacridine and acridine orange;
N-(p-(2-benzoxazolyl)phenyl)maleimide; cyanines, such as
indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5),
indodicarbocyanine 5.5 (Cy5.5),
3-(-carboxy-pentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine (CyA);
1H,5H-11H,15H-Xantheno[2,3,4-ij:5,6,7-i'j']diquinolizin-18-ium,
9-[2(or
4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or
2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or
Texas Red); and boron-dipyrromethene class (BODIPY) dyes. Other
detection labels may include the use of micro particles such as
quantum dots, nanoparticles, such as gold nanoparticles, and
microbeads. Alternatively, the detection molecule, label or tag may
include detection systems using multiple compounds (system which
requires the interaction of an additional independent compound),
such as a biotin-streptavidin system in which the biotin attaches
to the base and streptavidin is added separately. The detection
molecule, label or tag may include radioactive labels.
Compounds such as shown in FIG. 9N, FIG. 9O, and other similar
chromophore bearing modified deuterated nucleoside phosphoramidite
can be incorporated in one or more positions of the deuterated
oligonucleotide of FIG. 9H or 9F. One approach for the synthesis of
such deuterated oligonucleotide may include a protected primary
amine function being introduced at the end of a spacer molecule.
After introduction of the spacer, a deprotection step is carried
out to release the primary amine function, which subsequently is
reacted with various chromphore active ester and labeling reagents.
A variety of chromophores and ligands for nucleic acid modification
can be used, such as a C-6 amino modifier, CLP-1132, 5'-amino
modifier (MMT) modifier--C-12 spacer phosphoramidite, CLP-1585;
5'-Amino (TFA) Modifier--C12 spacer phosphoramidite; CLP-1575,
Biotin (BB) phosphoramidite, CLP-1517; biotin (BB) CPG, Biotin (BB)
TEG phosphoramidite, CLP-1518, symmetrical braching
phosphoramidite, CLP-5215 cholesterol TEG (tetraethyleneglycol)
phosphoramidite, (+)-alpha-tocopherol phosphoramidite, CLP-2706,
each commercially available from ChemGenes Corporation (Wilmington,
Mass.
FIG. 9G illustrates the deuterated oligonucleotide illustrated in
FIG. 8 modified to include at least one ligand or conjugate, i.e. a
substance that forms a complex or binds to at least a portion of
the modified, deuterated ribo-oligonucleotide. The ligand or
conjugate L may include, but is not limed to: (1) cyanoethyl
phosphate-polyethylene glycols.sup.v, where v is number of glycol
units, and can at least one 1, or preferably 2-100 glycol units;
(2) cyanoethyl phosphate-linker attached with cholesterol, biotin,
fluoresceins, cyanine dyes, psoralen, tetramethylrhodamine dye,
dabcyl dye, C-3 disulfide, C-6 disulfide, symmetrical and
asymmetrical hydrocarbon chain (C.sub.2-C.sub.50), symmetrical and
asymmetrical hydrocarbon chain (C.sub.2-C.sub.50) with a terminal
amino group protected with CF3C(.dbd.O) or phthalamido or FMOC,
(C.sub.1-C.sub.16)alkylene-amine protected with a amine protecting
group, (C.sub.1-C.sub.5)alkylene-amine protected with an azide
group; (C.sub.1-C.sub.5)alkylene-amine protected amine protected
with an acetylene (C triple bond CH) group; (3) cyanoethyl
phosphate-ethane-2-ol-protected with DMT group or other acid labile
groups, cyanoethyl phosphate-propane-3-ol-protected with DMT group
or other acid labile groups, (4) (C.sub.1-C.sub.50)alkylene with a
terminal hydroxy; (5) lipids, carboxyl groups, or peptides; or (6)
a branched phosphoramidite. The addition of ligand or conjugate L
may be useful in creating deuterated oligonucleotides which may
have better cellular uptake characteristics. Deuterated
oligonucleotides bearing a terminal lipophilic molecules or
lipophillic groups, (palmitoyl, cholesterol, oligonucleotide/Lipid
Particles stabilized with polyethyleneglycol, lipid molecules with
negatively charged albumin, polyethyleneglycol, lipid molecules
with negatively charged albumin distally attached to bilayer
anchored polyethylene glycol (PEG) chains, polyethylene PEG bearing
coated cationic lipoplexes (CCLs), PEG and keyhole limpet
hemocyanin) attached through a biodegradable function may be useful
as a prodrug with improved cellular uptake. FIG. 9H illustrates the
deuterated oligonucleotide of FIG. 9G having one or more detection
molecule, label or tag X1, X2, X3 as described above.
Referring to FIG. 9I, illustrates the deuterated oligonucleotide
shown in FIG. 8 having a deuterated deoxyribose (2-deoxyribose) end
cap attached to the end of the oligonucleotide. Inclusion of the
deoxyribose (2-deoxyribose) end cap provides the modified
deuterated oligonucleotide with increased stability, practically
towards stability against digestion with ribonucleases. As shown in
the figure, B1 refers to the bases associated with the deuterared
ribose units and B2 refers to the bases associated with the
deoxyribose, or any other base as descried previously. As
illustrated, the deoxyribose (2-deoxyribose) end cap includes two
deoxyribose units. However, the end cap may include n2 units, where
n is the number of deoxyribose based nucleotides or nucleosides,
such as for example, 1-200, or 2-100. The deoxyribose based
nucleotides or nucleosides forming the end cap may include natural
nucleobases, such as adenine, thymine, cytosine, guanine, or any
modified nucleobase. Preferably, the nucleobase is thymidine,
either deuterated or undeuterated thymadine. Each of the modified
structures shown in FIGS. 9E, 9F, 9G, 9H, and 9I, may further be
modified to include one or more 5 prime to 5 prime or three prime
to three prime linkage as described above for structures shown in
FIGS. 9C and 9D.
Synthesis of the deuterated oligonucleotide illustrated in FIG. 9I
may be accomplished using synthesis schemes described herein with
modifications. Synthesis of structure 9P-4 may be accomplished from
deuterated thymidine attached to a solid support as the first
3'-terminal deuterated thymidine nucleoside or as thymidine
3'-phosphoramidite, structure 9P-4, when there is second or
additional thymidine residues in the oligonucleotide, see FIG. 9P.
Selective introduction of 5'-DMT can be achieved on deuterated
thymidine structure 9P-1 to lead to 5'-DMT-deuterated thymidine
(structure 9P-2) (structure 9P-1 to structure 9P-2). Introduction
of a succinyl group at 3'-position can be accomplished in standard
reaction conditions to lead to solid support added
DMT-deuterereated thymidine (structure 9P-4). Similarly a
3'-phosphoramidite function can be introduced on compound
(structure 9P-2) to lead to 5'-DMT-deuterated
thymidine-3'-phosphoramidite (structure 9P-3).
As illustrated in FIG. 9Q-A, the general structure has been
modified so the deuterated oligonucleotide has one bond between
nucleosides including a 2 prime to 5 prime linkage. A shown in the
figure, the phosphate group (OWPYO) interlinks the ribose sugars.
In this case, the phosphate group bonded to the 2 prime carbon of a
ribose sugar unit interlinks to the next ribose sugar unit by
bonding to the 5 prime carbon. The general structure may also
include detecting molecules X1, X2, and X3, see FIG. 9Q-B. While
the modified oligonucleotide is shown having a multiple 2 prime to
5 linkages between, the oligonucleotide may contain only one 2
prime to 5 prime linkage and/or the 2 prime to 5 prime linkage can
be between any two ribose units. A 2 prime, 5 prime-linked
deuterated oligonucleotide may 1) provide stability, 2) act as
antivirals, antitumorals and/or immunomodulators or as potentiating
agents for interferon action, or 3) provide strong thermal
stability and antisense inhibition of gene expression. The
synthesis of deuterated oligonucleotides illustrated in FIG. 9Q-A
or FIG. 9Q-B can be accomplished using schemes described
previously, with modifications. The synthesis of oligonucleotides
of the general formula of FIG. 9R possessing deuterium modified
2',5'-linked oligonucleotides can be achieved from nucleosides
possessing 5'-DMT-3'-tBDMS-2'-phosphoramidites general structure,
structure 9S-3, see FIG. 9S. The general scheme for the synthesis
of this class of deuterium oligonucleotides are proposed to be
synthesized by the following scheme: (i) introduction of tBDMS
protecting group under known conditions to result in 2' and 3'
mixture of isomeric tBDMS nucleosides, structure 9S-2 and structure
9S-3, and (ii) Phosphitylation step under known reaction
conditions.
TABLE-US-00004 TABLE 3 Additional Deuterated/Natural
Oligonucleotide sequences. SEQUENCED IDEN- SEQUENCE TIFICATION
NUMBER (5' to 3') SEQ ID NO. 13 UCCCAUUUCCCU SEQ ID NO. 14
UGACAACAUUGU SEQ ID NO. 15 CUUCCCGAACAU SEQ ID NO. 16 ACACCACUUCCG
SEQ ID NO. 17 CGGAGGGCGGAU SEQ ID NO. 18 AUCAUCCGUGCU SEQ ID NO. 19
AAAUCCAAGUCA SEQ ID NO. 20 AGUUCAUGGUUU SEQ ID NO. 21 CUGAACCUGCAU
SEQ ID NO. 22 GUGCUUCAUUAU SEQ ID NO. 23 CCAGACUCGCAU SEQ ID NO. 24
CUCCAUUUUCAA SEQ ID NO. 25 GGAACAUGAGGC SEQ ID NO. 26 GGACGCUCACAU
SEQ ID NO. 27 UGAGCCAAAACA SEQ ID NO. 28 ACCAGUAGCAGC SEQ ID NO. 29
AUAAUCUUUCAU SEQ ID NO. 30 GAUAAGGACGAC SEQ ID NO. 31 GACCCAACAAAC
SEQ ID NO. 32 UGUGAUUCGGAA SEQ ID NO. 33 GAAGUAGGCAAA SEQ ID NO. 34
AGCUUUCAUCCU SEQ ID NO. 35 UUCUUCUGAAAUCAU SEQ ID NO. 36
UAAUAUAUCUGACAU SEQ ID NO. 37 UCCGGUUGAUGGCAU SEQ ID NO. 38
CAUUAUUUAAAGCAU SEQ ID NO. 39 UUGUUGUAUUUC SEQ ID NO. 40
GCGGUGUGUUUG SEQ ID NO. 41 CUCCUUUUUGGAUU SEQ ID NO. 42
UUUCAUACUUUUUC
Using any procedure outlined herein, oligonucleotide having SEQ ID
NO: 13-SEQ ID NO: 51 may be synthesized. SEQ ID NO: 13-SEQ ID NO:
51 may comprise partially or fully deuterated ribose structures,
and interlinked with a natural phosphodiester backbone or
phosphorothioate. Oligonucleotides having SEQ ID NO: 13-SEQ ID NO:
51 may include 3' to 3' prime linkages, such as illustrated in FIG.
9C, 5' to 5' prime linkages, such as illustrated in FIG. 9D, one or
more detection member(s), label(s), or tag(s), such as illustrated
in FIG. 9E, one or more ligands, such as illustrated in 9F, one
ligand and one or more detection members, labels, or tags; such as
illustrated in FIG. 9G, or having a deoxyribose end cap, such as
illustrated in FIG. 9H. Additionally, oligonucleotides having SEQ
ID NO: 13-SEQ ID NO: 174 may be synthesized, using deuterated
phosphoramidites and nature phosphoramidites, having a mixture of
partially or fully deuterated ribose and natural ribose attached to
nucleobases, and a natural phosphodiester linkage, or variant
nucleotide linkages such as a phosphorothioate linkage.
All patents and publications mentioned in this specification are
indicative of the levels of those skilled in the art to which the
invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
It is to be understood that while a certain form of the invention
is illustrated, it is not to be limited to the specific form or
arrangement herein described and shown. It will be apparent to
those skilled in the art that various changes may be made without
departing from the scope of the invention and the invention is not
to be considered limited to what is shown and described in the
specification and any drawings/figures included herein.
One skilled in the art will readily appreciate that the present
invention is well adapted to carry out the objectives and obtain
the ends and advantages mentioned, as well as those inherent
therein. The embodiments, methods, procedures and techniques
described herein are presently representative of the preferred
embodiments, are intended to be exemplary and are not intended as
limitations on the scope. Changes therein and other uses will occur
to those skilled in the art which are encompassed within the spirit
of the invention and are defined by the scope of the appended
claims. Although the invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes for carrying out the invention which are obvious to
those skilled in the art are intended to be within the scope of the
following claims.
SEQUENCE LISTINGS
1
42112RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 1cucucucucu cu 12215RNAArtificial
SequenceDescription of Artificial Sequence Synthesized nucleic acid
molecule 2cauugguuca aacau 15312RNAArtificial SequenceDescription
of Artificial Sequence Synthesized nucleic acid molecule
3agguucaaac au 12415RNAArtificial SequenceDescription of Artificial
Sequence Synthesized nucleic acid molecule 4cauugguuca aacau
15512RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 5uugaugaaac au 12612RNAArtificial
SequenceDescription of Artificial Sequence Synthesized nucleic acid
molecule 6caguucaaac au 12715RNAArtificial SequenceDescription of
Artificial Sequence Synthesized nucleic acid molecule 7gaccaguuca
aacau 15812RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 8agguucaaac au 12912RNAArtificial
SequenceDescription of Artificial Sequence Synthesized nucleic acid
molecule 9aaacgccucc au 121015RNAArtificial SequenceDescription of
Artificial Sequence Synthesized nucleic acid molecule 10aaaugaaaau
gucau 151112RNAArtificial SequenceDescription of Artificial
Sequence Synthesized nucleic acid molecule 11aaauucuaac au
121215RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 12uucaaauucu aacau
151312RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 13ucccauuucc cu
121412RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 14ugacaacauu gu
121512RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 15cuucccgaac au
121612RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 16acaccacuuc cg
121712RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 17cggagggcgg au
121812RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 18aucauccgug cu
121912RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 19aaauccaagu ca
122012RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 20aguucauggu uu
122112RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 21cugaaccugc au
122212RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 22gugcuucauu au
122312RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 23ccagacucgc au
122412RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 24cuccauuuuc aa
122512RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 25ggaacaugag gc
122612RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 26ggacgcucac au
122712RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 27ugagccaaaa ca
122812RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 28accaguagca gc
122912RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 29auaaucuuuc au
123012RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 30gauaaggacg ac
123112RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 31gacccaacaa ac
123212RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 32ugugauucgg aa
123312RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 33gaaguaggca aa
123412RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 34agcuuucauc cu
123515RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 35uucuucugaa aucau
153615RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 36uaauauaucu gacau
153715RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 37uccgguugau ggcau
153815RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 38cauuauuuaa agcau
153912RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 39uuguuguauu uc
124012RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 40gcgguguguu ug
124114RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 41cuccuuuuug gauu
144214RNAArtificial SequenceDescription of Artificial Sequence
Synthesized nucleic acid molecule 42uuucauacuu uuuc 14
* * * * *